Chem. Rev. 2002, 102, 4501−4523
4501
Serine Protease Mechanism and Specificity
Lizbeth Hedstrom
Department of Biochemistry, MS 009, Brandeis University, Waltham, Massachusetts 02454
Received May 14, 2002
Contents
I. Introduction
II. The Structure of Chymotrypsin-Like Serine
Proteases
A. The Catalytic Components
1. The Catalytic Triad
2. The Oxyanion Hole
B. The Substrate Recognition Sites
1. The S1 Site
2. The Polypeptide Binding Site
3. The S2−Sn Sites
4. The S1ʹ′−S3ʹ′ Sites
C. The Zymogen Activation Domain
III. The Mechanism of Serine Proteases
A. The Generally Accepted Chemical
Mechanism of Serine Protease Catalysis
1. The Discovery of the Catalytic Triad and
the Oxyanion Hole
2. Mutagenesis of the Catalytic Triad
3. Evidence for the Acylenzyme Intermediate
4. Evidence for the Tetrahedral Intermediate
B. Mechanisms of Inactivation and Inhibition
1. Transition State Analogue Inhibitors
2. Stable Acylenzymes
C. Issues in the Function of the Catalytic Triad
1. Where Are the Protons? One-Proton
versus Two-Proton-Transfer Mechanisms
2. Low Barrier Hydrogen Bonds
3. Moving His Mechanisms
4. The Hydrolytic Water
5. Stereochemistry of the Tetrahedral
Intermediate
6. Stabilization of the Tetrahedral
Intermediate/Strain in the Acylenzyme
Intermediate
IV. Kinetics of the Serine Protease Reaction
V. Substrate Discrimination during Catalysis
A. The Hallmarks of Serine Protease Specificity
B. Specificity Is Determined by the Binding and
Acylation Steps
C. The Contribution of Substrate Association to
Specificity
D. Implications for the Specificity of Ester
Hydrolysis
VI. Redesigning the Trypsin-Chymotrypsin-Elastase
Paradigm
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VII.
VIII.
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* E -m a il: h edst r om @br a n deis.edu ; ph on e: 781-736-2333; F AX:
781-736-2349.
IX.
X.
XI.
A. Redesigning Amidase Activity
B. Redesigning Esterase Activity
How Do Remote Interactions Translate into
Catalysis?
A. Remote Interactions Align the Substrate in
the Active Site
B. Remote Interactions Are Optimized in the
Transition State
C. Remote Interactions Induce a Conformational
Change that Favors Catalysis
D. The Remote Interactions Shield the Catalytic
Triad from Solvent
E. Remote Interactions Couple Catalysis to
Motion of the Protease Structure
Specificity via Induced Fit and Allostery
A. Induced Fit as a Mechanism for Narrow
Specificity
B. Factor D: An Induced Fit Protease
C. Alpha Lytic Protease: Induced Fit as a
Mechanism for Broad Specificity
How Serine Proteases Work: Summary and
Prospects
Useful Web Sites
References
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I. Introduction
Alm ost on e-t h ir d of a ll pr ot ea ses ca n be cla ssified
a s ser in e pr ot ea ses, n a m ed for t h e n u cleoph ilic Ser
r esidu e a t t h e a ct ive sit e. Th is m ech a n ist ic cla ss wa s
or igin a lly dist in gu ish ed by t h e pr esen ce of t h e AspH is-Ser “ch a r ge r ela y” syst em or “ca t a lyt ic t r ia d”. 1
Th e Asp-H is-Ser t r ia d ca n be fou n d in a t lea st fou r
differ en t st r u ct u r a l con t ext s, in dica t in g t h a t t h is
ca t a lyt ic m a ch in er y h a s evolved on a t lea st fou r sepa r a t e occa sion s.2 Th ese fou r cla n s of ser in e pr ot ea ses
a r e t ypified by ch ym ot r ypsin , su bt ilisin , ca r boxypept ida se Y, a n d Clp pr ot ea se (ME ROP S n om en cla t u r e;3
Ta ble 1). Mor e r ecen t ly, ser in e pr ot ea ses wit h n ovel
ca t a lyt ic t r ia ds a n d dya ds h a ve been discover ed,
including Ser-His-Glu, Ser-Lys/His, His-Ser-His,
a n d N-t er m in a l Ser .2 Sever a l of t h ese n ovel ser in e
pr ot ea ses a r e su bject s of a ccom pa n yin g a r t icles in
t h is issu e.
Th is a r t icle will r eview r ecen t wor k on t h e m ech a n ism a n d specificit y of ch ym ot r ypsin -like en zym es,
wit h t h e occa sion a l r efer en ces t o per t in en t exper im en t s wit h su bt ilisin . Ch ym ot r ypsin -like pr ot ea ses
a r e t h e m ost a bu n da n t in n a t u r e, wit h over 240
pr ot ea ses r ecogn ized in t h e ME ROP S da t a ba se.3
10.1021/cr000033x CCC: $39.75 © 2002 American Chemical Society
Published on Web 11/23/2002
4502 Chemical Reviews, 2002, Vol. 102, No. 12
Hedstrom
Ta ble 1. S e rin e P ro te a s e Cla n s a
cla n
m em ber s
P A(S)
SB
SC
SK
301
91
64
14
SE
SF
SH
SM
SN
P B(S)
16
24
7
7
4
4
exa m ple
ca t a lyt ic r esidu es
dist r ibu t ion
“Cla ssic” Ca t a lyt ic Tr ia d Ser in e P r ot ea ses
ch ym ot r ypsin
H is-Asp-Ser
su bt ilisin
Asp-H is-Ser
ca r boxypept ida se Y
Ser -Asp-H is
Clp pr ot ea se
Ser -H is-Asp
B,
B,
B,
B,
Ar ,
Ar ,
Ar ,
Ar ,
“Novel” Ser in e P r ot ea ses
ca r boxypept ida se A
sign a l pept ida se I
cyt om ega lovir u s a ssem blin
C-t er m in a l pr ocessin g pr ot ea se-1
dipept ida se E
pen icillin a m idoh ydr ola se pr ecu r sor
B,
B,
V
B,
B,
B,
Ar , P l, An
Ar , F , P l, An , V
D -Ala -D -Ala
Ser -Lys
Ser -Lys/H is
H is-Ser -H is
Ser -Lys
Ser -H is-Glu
N-t er m in a l Ser
F , P l, An , V
P r , F ,P l,An ,V
P r , F , P l, An ,
P r , F , P l,An ,V
Ar , P l
An
Ar , P r
a
Th e ME ROP S da t a ba se cla ssifies pr ot ea ses ba sed on st r u ct u r a l sim ila r it y [Ra wlin gs, N. D., a n d Ba r r et t , A. J . (2000) ME ROP S:
Th e P ept ida se Da t a ba se. N u cleic Acid s R es. 28, 323-325]. Th e followin g in for m a t ion is t a bu la t ed fr om ME ROP S r elea se 5.7
[h t t p://m er ops.ia pc.bbsr c.a c.u k/]. Ca t a lyt ic r esidu es a r e list ed in t h e or der t h a t t h ey a ppea r in t h e pr im a r y sequ en ce. B, ba ct er ia ;
Ar , a r ch a ea ; Az, a r ch ezoa ; P r , pr ot ozoa ; F , fu n gi; P l, pla n t s; An , a n im a ls; V, vir u ses. In a ddit ion t o t h e pr ot ea ses list ed below, 21
ser in e pr ot ea ses h a ve been iden t ified t h a t a r e n ot yet a ssign ed t o a cla n .
These proteases can be found in eukaryotes, prokaryot es, a r ch a e, a n d vir u ses. Ch ym ot r ypsin -like pr ot ea ses a r e in volved in m a n y cr it ica l ph ysiologica l
pr ocesses, in clu din g digest ion , h em ost a sis, a popt osis,
sign a l t r a n sdu ct ion , r epr odu ct ion , a n d t h e im m u n e
r espon se (Ta ble 2).4-8 “Ca sca des” of sequ en t ia l a ct iva t ion of ser in e pr ot ea ses dr ive blood coa gu la t ion ,
com plem en t fixa t ion , a n d fibr in olysis.9-11 Sim ila r
pr ot ea se ca sca des a ppea r t o be in volved in developm en t , m a t r ix r em odelin g, differ en t ia t ion , a n d wou n d
h ea lin g.12-14 Th ese diver se ph ysiologica l n ich es demand proteases with wildly varied specificities, rangin g fr om digest ive pr ot ea ses t h a t clea ve a ft er h ydr oph obic or posit ively ch a r ged r esidu es, t o pr ot ea ses
t h a t r ecogn ize a five-r esidu e clea va ge sit e or even a
sin gle pr ot ein . Ser in e pr ot ea se specificit y ca n u su a lly
be r a t ion a lized by t h e t opology of t h e su bst r a t e
bin din g sit es a dja cen t t o t h e ca t a lyt ic t r ia d (t h e
“a ct ive sit e cleft ”). Sever a l excellen t r eviews h a ve
Dr. Lizbeth Hedstrom was born in Frederick, Maryland, in 1958. She
obtained a Bachelor of Science in Chemistry at the University of Virginia
and then moved to Brandeis University where she obtained a Ph.D. in
Biochemistry under the direction of Robert Abeles in 1985. After
postdoctoral work at the University of California San Francisco with C. C.
Wang and William Rutter, Dr. Hedstrom returned to Brandeis University
in 1992. She is currently the Markey Associate Professor of Biochemistry.
Her research interests include structure/function relationships in enzyme
catalysis, protein engineering, the design of novel enzyme inhibitors as
mechanistic probes, and potential pharmaceutical agents. Her work
currently focuses on serine proteases and enzymes involved nucleotide
metabolism.
Ta ble 2. Re p re s e n ta tiv e Ma m m a lia n
Ch y m o try p s in -Lik e P ro te a s e s
digest ive pr ot ea ses
ch ym ot r ypsin
t r ypsin
pa n cr ea t ic ela st a se
im m u n e r espon se
t r ypt a se
com plem en t fa ct or D
com plem en t fa ct or B
com plem en t fa ct or C
com plem en t com pon en t 2
m a st cell pr ot ea se
ca t h epsin G
n eu t r oph il ela st a se
blood coa gu la t ion
coa gu la t ion fa ct or VIIa
coa gu la t ion fa ct or IXa
coa gu la t ion fa ct or Xa
coa gu la t ion fa ct or XIIa
t h r om bin
pr ot ein C
fibr in olysis
u r okin a se
t issu e pla sm in ogen a ct iva t or
pla sm in
ka llikr ein
r epr odu ct ion
pr ost a t e specific a n t igen
a cr osein
ba ct er ia l h om ologu es
a lph a lyt ic pr ot ea se
S treptom yces griseu s pr ot ea se A
S treptom yces griseu s pr ot ea se B
r ecen t ly r epor t ed on t h is t opic. 15-17 H owever , wh ile
t h is per spect ive im plies t h a t specificit y ca n be r edu ced t o discr im in a t ion in su bst r a t e bin din g, specificit y is pr im a r ily expr essed in t h e r a t es of ch em ica l
t r a n sfor m a t ion . Th is r eview will discu ss t h e expr ession of specificit y du r in g ca t a lysis a n d st r a t egies for
su bst r a t e discr im in a t ion , focu sin g on t h e ch ym ot r ypsin -t r ypsin -ela st a se pa r a digm a n d in clu din g
exa m ples fr om F a ct or D, a lph a lyt ic pr ot ea se, a n d
t h r om bin .
II. The Structure of Chymotrypsin-Like Serine
Proteases
F ir st we will con sider t h e st r u ct u r e of ch ym ot r ypsin -like pr ot ea ses (F igu r e 1). Ch ym ot r ypsin h a s
Serine Protease Mechanism and Specificity
Chemical Reviews, 2002, Vol. 102, No. 12 4503
in t er t win ed. In a ddit ion t o t h e pr ot ea se dom a in ,
m a n y ser in e pr ot ea ses a r e m odu la r pr ot ein s con t a in in g a u xilia r y r egu la t or y dom a in s (e.g., kr in gle, E GF ,
et c.).20 Th e fu n ct ion s of t h ese a u xilia r y dom a in s will
n ot be con sider ed h er e.
A. The Catalytic Components
1. The Catalytic Triad
F ig u re 1. St r u ct u r e of ch ym ot r ypsin . Th is figu r e wa s
pr odu ced wit h Molscr ipt v2.1 (r ef 267) a n d P OV-Ra y by
An n et t e P a st er n a k.
245 r esidu es a r r a n ged in t wo six-st r a n ded bet a
ba r r els.18 Th e a ct ive sit e cleft is loca t ed bet ween t h e
t wo ba r r els. Th is st r u ct u r e is gen er a lly divided in t o
ca t a lyt ic, su bst r a t e r ecogn it ion a n d zym ogen a ct iva t ion dom a in com pon en t s t h a t a r e com m on t o a ll
ch ym ot r ypsin -like ser in e pr ot ea ses. (Th e “loop 1”
n om en cla t u r e u sed in som e pr eviou s pa per s a n d
r eviews will be r epla ced wit h a syst em ba sed on
ch ym ot r ypsin ogen n u m ber in g t o fa cilit a t e com pa r ison a m on g differ en t pr ot ea ses.) Th is or ga n iza t ion is
derived from Kraut 19 and is convenient for discussion,
but wrongly implies that catalysis, substrate recognit ion , a n d zym ogen a ct iva t ion a r e sepa r a t e pr ocesses.
As will be seen , t h ese t h r ee pr ocesses in volve m a n y
of t h e sa m e st r u ct u r a l fea t u r es a n d a r e in t r ica t ely
Th e ca t a lyt ic t r ia d spa n s t h e a ct ive sit e cleft , wit h
Ser 195 on on e side a n d Asp102 a n d H is57 on t h e
ot h er (F igu r e 1). H is57 a ssu m es t h e less fa vor a ble
Nδ1-H t a u t om er (F igu r e 2), in dica t in g t h a t t h e
tautomer equilibrium is perturbed at least 100× from
t h e solu t ion posit ion .21 Th e ca t a lyt ic t r ia d is pa r t of
a n ext en sive h ydr ogen bon din g n et wor k (F igu r e 2A).
H ydr ogen bon ds a r e gen er a lly obser ved bet ween t h e
Nδ1-H of H is57 a n d Oδ1 of Asp102 a n d bet ween t h e
OH of Ser 195 a n d t h e N!2-H of H is57, a lt h ou gh t h e
la tter hydrogen bond is lost when His57 is protonated
(F igu r e 2). Sim ila r h ydr ogen bon ds a r e obser ved in
protein inhibitor (eglin C), acylenzyme, and transition
st a t e a n a logu e (t r iflu or oket on e) com plexes, wh ich
su ggest s t h a t t h ese h ydr ogen bon ds a r e pr esen t
t h ou gh ou t t h e ca t a lyt ic cycle. In t er est in gly, t h e
H is57-Asp102 h ydr ogen bon d h a s t h e syn or ien t a t ion r ela t ive t o t h e ca r boxyla t e, so t h a t t h e h ydr ogen
bon d for m s wit h t h e m or e ba sic elect r on pa ir .22,23 Th e
OH of Ser 214 for m s a h ydr ogen bon d wit h Oδ1 of
Asp102 in a lm ost a ll ch ym ot r ypsin -like pr ot ea ses.
Ser 214 wa s on ce con sider ed t h e fou r t h m em ber of t h e
ca t a lyt ic t r ia d, a lt h ou gh m or e r ecen t eviden ce in dica t es t h a t it is dispen sa ble.24,25 H ydr ogen bon ds a r e
a lso obser ved bet ween t h e Oδ2 of Asp102 a n d t h e
m a in ch a in NH s of Ala 56 a n d H is57. Th ese h ydr ogen
F ig u re 2. H ydr ogen bon din g in t h e ca t a lyt ic t r ia d. (A) Th e ca t a lyt ic t r ia d in ch ym ot r ypsin com plexes. Th e h ydr ogen
bon din g n et wor k of t h e ca t a lyt ic t r ia d is depict ed for t h e com plex wit h a pr ot ein in h ibit or eglin C, a n a cylen zym e
in t er m edia t e a n d a t r iflu or oket on e t r a n sit ion st a t e a n a logu e. Th e dot t ed lin es r epr esen t pot en t ia l h ydr ogen bon ds a n d
t h e n u m ber s a r e t h e dist a n ces bet ween h et er oa t om s in Å. (B) Th e effect of t h e C!1-H h ydr ogen bon d on ch a r ge dist r ibu t ion
in H is57. In t h e a bsen ce of t h e h ydr ogen bon d, t h e posit ive ch a r ge will a ccu m u la t e a t Nδ1 wh er e it ca n be st a bilized by
Asp102.
4504 Chemical Reviews, 2002, Vol. 102, No. 12
bon ds a r e believed t o or ien t Asp102 a n d H is57. In
a ddit ion , a n ovel h ydr ogen bon d is obser ved bet ween
t h e C!1-H of H is57 a n d t h e m a in ch a in ca r bon yl of
Ser 214.26,27 Sim ila r h ydr ogen bon ds a r e a lso obser ved
in su bt ilisin a n d ot h er cla sses of ser in e h ydr ola ses,
su ggest in g t h a t t h is in t er a ct ion m a y be im por t a n t for
ca t a lyt ic t r ia d fu n ct ion .26 Th is h ydr ogen bon d m a y
pr even t t h e loca liza t ion of posit ive ch a r ge on Nδ1 of
H is57 (F igu r e 2B). In t er est in gly, t h e ca r bon yl oxygen
of Ser 214 is a lso pa r t of t h e polypept ide bin din g sit e
(see below), a n d m a y m edia t e com m u n ica t ion bet ween su bst r a t e a n d t h e ca t a lyt ic t r ia d.
2. The Oxyanion Hole
Th e oxya n ion h ole is for m ed by t h e ba ckbon e NH s
of Gly193 a n d Ser 195. Th ese a t om s for m a pocket of
posit ive ch a r ge t h a t a ct iva t es t h e ca r bon yl of t h e
scissile pept ide bon d a n d st a bilizes t h e n ega t ively
charged oxyanion of the tetrahedral intermediate (see
below). Th e oxya n ion h ole is st r u ct u r a lly lin ked t o
t h e ca t a lyt ic t r ia d a n d t h e Ile16-Asp194 sa lt br idge
via Ser 195. Su bt ilisin a lso con t a in s a n oxya n ion h ole,
for m ed by t h e side ch a in s of Asn 155, Th r 220, a n d t h e
ba ckbon e NH of Ser 221.28
Hedstrom
for a cidic P 1 r esidu es is a t t r ibu t ed t o Ar g226.35 Th ese
obser va t ion s su ggest t h a t a sm a ll set of st r u ct u r a l
elem en t s con t r ols t h e specificit y of t h ese pr ot ea ses.
H owever , a s det a iled below, sim ple t r a n sposit ion of
these structural elements does not switch S1 specificit y.
2. The Polypeptide Binding Site
Protea se-su bstra te in tera ction s exten d beyond t he
S1 sit e, m in im a lly in clu din g t h e polypept ide bin din g
sit e a n d oft en in volvin g a ddit ion a l bin din g pocket s.
Th e polypept ide bin din g sit e r efer s t o t h e m a in ch a in
of r esidu es 214-216 wh ich for m a n a n t ipa r a llel bet a
sh eet wit h t h e ba ckbon e of t h e P 1-P 3 r esidu es of a
pept ide su bst r a t e (F igu r e 3). In ch ym ot r ypsin , h y-
B. The Substrate Recognition Sites
The substrate recognition sites include the polypept ide bin din g sit e a n d t h e bin din g pocket s for t h e side
ch a in s of t h e pept ide su bst r a t e. In vest iga t ion s of
pr ot ea se specificit y h a ve gen er a lly focu sed on t h e
P 1/S1 in t er a ct ion (ba sed on t h e n om en cla t u r e of
Sch ech t er a n d Ber ger (r ef 29), wh er e P 1-P 1ʹ′ den ot es
pept ide r esidu es on t h e a cyl a n d lea vin g gr ou p side
of the scissile bond, respectively. The adjacent peptide
r esidu es a r e n u m ber ed ou t wa r d, a n d S1, S1ʹ′, et c.
den ot e t h e cor r espon din g en zym e bin din g sit es),
followed by con sider a t ion of t h e P 2-P n /S2-Sn in teractions. The S1ʹ′-Snʹ′ sites are generally overlooked
du e t o t h e pr eva len ce of a ssa ys u sin g su bst r a t es wit h
ch r om oph or ic lea vin g gr ou ps. We will con sider t h e
S1 sit e, t h e polypept ide bin din g sit e, t h e S2-Sn a n d
S1ʹ′-Sn ʹ′ sit es sepa r a t ely.
1. The S1 Site
Specificit y of ch ym ot r ypsin -like ser in e pr ot ea ses is
u su a lly ca t egor ized in t er m s of t h e P 1-S1 in t er a ct ion . Th e S1 sit e is a pocket a dja cen t t o Ser 195,
formed by residues 189-192, 214-216, and 224-228.
Specificit y is u su a lly det er m in ed by t h e r esidu es a t
posit ion s 189, 216, a n d 226 (r eviewed in r efs 15 a n d
17). F or exa m ple, t h e specificit y of ch ym ot r ypsin
cor r ela t es wit h t h e h ydr oph obicit y of t h e P 1 r esidu e,
wit h P 1-P h e pr efer r ed over Ala by 50000.30,31 Th e
com bin a t ion of Ser 189, Gly216, a n d Gly226 cr ea t e a
deep h ydr oph obic pocket in ch ym ot r ypsin t h a t a ccou n t s for t h is specificit y.18 Asp189, Gly216, a n d
Gly226 cr ea t e a n ega t ively ch a r ged S1 sit e t h a t
a ccou n t s for t r ypsin ’s specificit y for su bst r a t es con t a in in g Ar g or Lys a t P 1.32,33 E la st a se pr efer s su bst r a t es wit h sm a ll a liph a t ic r esidu es a t P 1; t h e S1
sit e of ela st a se is sm a ller t h a n t h e S1 sit es of
ch ym ot r ypsin a n d t r ypsin du e t o t h e pr esen ce of
Va l216 a n d Th r 226.34 Th e specificit y of gr a n zym e B
F ig u re 3. Su bsit e bin din g of por cin e pa n cr ea t ic a n d
h u m a n leu kocyt e ela st a se. Th e h ydr ogen bon din g in t er a ct ion of t h e polypept ide bin din g sit e a n d Sʹ′ sit es a r e sh own
by dot t ed lin es. Th e r esidu es t h a t for m ea ch su bsit e a r e
list ed. Modified fr om r ef 53.
dr ogen bon ds for m bet ween t h e ca r bon yl oxygen of
Ser 214 a n d t h e NH of t h e P 1 r esidu e, t h e NH of
Tr p215 a n d t h e ca r bon yl of P 3 a n d t h e ca r bon yl of
Gly216 a n d t h e NH of P 3. Th ese in t er a ct ion s a r e a
gen er a l fea t u r e of ch ym ot r ypsin -like pr ot ea ses a n d
a r e cr it ica l for efficien t su bst r a t e h ydr olysis. In t er est in gly, Gly216 h a s differ en t con for m a t ion s in ch ym ot r ypsin , t r ypsin , a n d ela st a se, 16 wh ich su ggest s
t h a t t h e st r en gt h of t h is h ydr ogen bon d will va r y.
Not e t h a t r esidu es 214-216 a lso for m on e wa ll of t h e
S1 sit e, a n d t h a t t h e ca r bon yl of Ser 214 for m s a
h ydr ogen bon d t o H is57. Th ese st r u ct u r a l in t er a ct ion s for m a lin e of com m u n ica t ion bet ween t h e
polypeptide bin din g site, th e S1 site, a n d th e ca t a lyt ic
t r ia d.
In terestin gly, th e in h ibitory loops of protein inhibit or s of ser in e pr ot ea ses a r e locked in t h e ext en ded
con for m a t ion r equ ir ed t o for m t h e a n t ipa r a llel bet a
sh eet wit h t h e polypept ide bin din g sit e. Th is ext en ded con for m a t ion , t er m ed t h e “ca n on ica l con for m a t ion ”, is obser ved in n u m er ou s pr ot ea se-in h ibit or
com plexes, in clu din g t r a n sit ion st a t e a n a logu es a n d
pept idyl a cylen zym es.36-45 Th e ca n on ica l con for m a t ion in clu des t h e P 3-P 3ʹ′ r esidu es a n d ca n ext en d t o
th e a m ide NH of residu e 218 a n d th e ca rbonyl oxygen
of P 5 a s obser ved in ela st a se (F igu r e 3). Im por t a n t ly,
t h e bet a sh eet st r u ct u r e ca u ses t h e side ch a in s t h e
pept ide su bst a t e t o poin t in a lt er n a t in g dir ect ion s.
Th e r eleva n ce of t h e a n t ipa r a llel bet a sh eet in t er a ct ion t o pept ide h ydr olysis h a s r ecen t ly been ca lled
in t o qu est ion beca u se disr u pt ion of t h ese h ydr ogen
Serine Protease Mechanism and Specificity
bon din g in t er a ct ion s h a d lit t le effect on su bst r a t e
h ydr olysis.46 H owever , t h e va lu es of K d for pr ot ein
in h ibit or s a n d t r a n sit ion st a t e a n a logu es cor r ela t e
wit h k ca t /K m for h ydr olysis of t h e a n a logou s su bst r a t es, wh ich a r gu es st r on gly t h a t su bst r a t es a lso
a ssu m e t h e ca n on ica l con for m a t ion .38,47-49
3. The S2−Sn Sites
Th e S2-S3 sit es of ch ym ot r ypsin displa y lit t le
su bst r a t e discr im in a t ion , in keepin g wit h ch ym ot r ypsin ’s fu n ct ion a s a digest ive pr ot ea se.50 Th e S2
sit e is a sh a llow h ydr oph obic gr oove bou n ded by
H is57, Tr p215, a n d Leu 99; t h e specificit y of t h e S2
sit e cor r ela t es wit h h ydr oph obicit y of t h e P 2 side
ch a in .49 Th e S3 sit e of ch ym ot r ypsin h a s lit t le specificit y, a n d ca n even a ccom m oda t e D -a m in o a cids; t h e
side ch a in of t h e P 3 r esidu e pr ot r u des ou t of t h e
a ct ive sit e cleft .
In ser t ion s in t h e loops su r r ou n din g t h e a ct ive sit e
cleft cr ea t e m or e defin ed pocket s for t h e S2-Sn sit es
of other serine proteases. These sites can be the major
determinant of specificity,51 as exemplifed by elastase,
wh er e t h e P 3/S3 in t er a ct ion is dom in a n t ,52,53 a n d
en t er okin a se (a lso kn own a s en t er opept ida se), wh ich
r ecogn izes t h e P 5-P 1 sequ en ce Asp-Asp-AspAsp-Lys wit h r est r ict ion en zym e-like pr ecision .54
4. The S1ʹ′−S3ʹ′ Sites
Th e in t er a ct ion s of t h e lea vin g gr ou p wit h t h e S1ʹ′Sn ʹ′ sit es a r e u su a lly in fer r ed fr om pr ot ein in h ibit or
st r u ct u r es t h a t do n ot con t a in t h e opt im u m P 1ʹ′-P n ʹ′
r esidu es. Th er efor e, t h e pr ecise in t er a ct ion s of t h e
P ʹ′ side ch a in s h a ve n ot been defin ed. A h ydr ogen
bon d bet ween t h e m a in ch a in NH of t h e P 2ʹ′ r esidu e
a n d t h e m a in ch a in ca r bon yl oxygen of P h e41 is a
con st a n t fea t u r e of t h e P ʹ′/Sʹ′ in t er a ct ion s. Ch ym ot r ypsin pr efer s su bst r a t es wit h P 1ʹ′-Ar g or Lys,
wh ich is a t t r ibu t ed t o elect r ost a t ic in t er a ct ion s wit h
Asp35 a n d Asp64.55 Th e P 1ʹ′ a n d P 3ʹ′ r esidu es poin t
in t h e sa m e dir ect ion a s a con sequ en ce of t h e bet a
sh eet a lign m en t of t h e su bst r a t e, so t h a t t h e S1ʹ′ a n d
S3ʹ′ sit es over la p. Th e S1ʹ′/S3ʹ′ sit es a r e bou n ded by
H is57, t h e 60’s loop a n d t h e 40’s loop. Th e P 2ʹ′ r esidu e
points in the opposite direction a nd ma y intera ct with
t h e 150’s loop. As a bove, in ser t ion s in t h ese loops ca n
cr ea t e m or e defin ed S1ʹ′-S3ʹ′ bin din g sit es.51
C. The Zymogen Activation Domain
Ch ym ot r ypsin -like pr ot ea ses a r e syn t h esized a s in a ctive precursors (“zymogens”) conta ining N-terminal
ext en sion s. F ou r segm en t s a r e defor m ed in t h e zymogens of chymotrypsin and trypsin: the N-terminus
t o r esidu e 19, r esidu es 142-152, 184-193, a n d 216223 (t h ese r egion s a r e collect ively t er m ed t h e a ct iva t ion dom a in 36 ). Th is defor m ed r egion in clu des t h e S1
sit e a n d oxya n ion h ole, wh ich expla in s t h e low
a ct ivit y of t h e zym ogen . P r ot eolyt ic pr ocessin g a ct iva t es t h e zym ogen , r elea sin g t h e N-t er m in a l Ile16.
Th e n ew N-t er m in u s for m s a bu r ied sa lt br idge wit h
Asp194, in du cin g a con for m a t ion a l ch a n ge t h a t or der s t h e a ct iva t ion dom a in . Th e S1 sit e a n d oxya n ion
h ole a r e for m ed, cr ea t in g t h e a ct ive pr ot ea se. Im por -
Chemical Reviews, 2002, Vol. 102, No. 12 4505
t a n t ly, t h e loss of pr ot ea se a ct ivit y a t h igh pH is
a t t r ibu t ed t o t h e depr ot on a t ion of t h e N-t er m in u s
a n d disr u pt ion of Ile16-Asp194 sa lt br idge, sh ift in g
t h e con for m a t ion a l equ ilibr iu m t owa r d a n in a ct ive
zym ogen -like con for m a t ion . 56,57 Th is m ech a n ism of
zym ogen a ct iva t ion is con ser ved a m on g m a m m a lia n
ch ym ot r ypsin -like ser in e pr ot ea ses. Ba ct er ia l h om ologu es h a ve a differ en t m ech a n ism of zym ogen a ct iva t ion , bu t r et a in a sa lt br idge in volvin g Asp194.58
III. The Mechanism of Serine Proteases
Obviou sly, a det a iled u n der st a n din g of t h e en zym a t ic r ea ct ion m ech a n ism is r equ ir ed t o u n der st a n d
h ow specificit y is gen er a t ed. F or t u n a t ely, ch ym ot r ypsin -like pr ot ea ses occu py a dist in gu ish ed pla ce
in t h e a n n a ls of bioch em ist r y a s per h a ps t h e best
st u died en zym e syst em .
A. The Generally Accepted Chemical Mechanism
of Serine Protease Catalysis
All pr ot ea ses m u st over com e t h r ee obst a cles t o
h ydr olyze a pept ide bon d: (a ) a m ide bon ds a r e ver y
st a ble du e t o elect r on don a t ion fr om t h e a m ide
n it r ogen t o t h e ca r bon yl. F or com pa r ison , a sim ple
a lkyl est er is ∼3000× m or e r ea ct ive t h a n a n a m ide
bon d, wh ile a p-n it r oph en yl est er is 300000× m or e
r ea ct ive.59 P r ot ea ses u su a lly a ct iva t e a n a m ide bon d
via t h e in t er a ct ion of t h e ca r bon yl oxygen wit h a
gen er a l a cid, a n d m a y a lso dist or t t h e pept ide bon d
t o disr u pt r eson a n ce st a biliza t ion ; (b) wa t er is a poor
n u cleoph ile; pr ot ea ses a lwa ys a ct iva t e wa t er , u su a lly
via a gen er a l ba se; a n d (c) a m in es a r e poor lea vin g
groups; protea ses protona te the a mine prior to expulsion . Ser in e pr ot ea ses per for m t h ese t a sks ver y
efficien t ly: t h e r a t es of pept ide h ydr olysis by ser in e
protea ses a re ∼10 10-fold greater than the uncatalyzed
r ea ct ion s. Obviou sly, t h ese m ech a n ism s of ca t a lysis
a r e n ot con fin ed t o pept ide h ydr olysis; ser in e pr ot ea ses a lso r ea dily h ydr olyze ot h er a cyl com pou n ds,
in clu din g a m ides, a n ilides, est er s, a n d t h ioest er s.
F igu r e 5 displa ys t h e gen er a lly a ccept ed m ech a n ism for ch ym ot r ypsin -like ser in e pr ot ea ses. In t h e
a cyla t ion h a lf of t h e r ea ct ion , Ser 195 a t t a cks t h e
ca r bon yl of t h e pept ide su bst r a t e, a ssist ed by H is57
a ct in g a s a gen er a l ba se, t o yield a t et r a h edr a l
in t er m edia t e. Th e r esu lt in g H is57-H + is st a bilized by
t h e h ydr ogen bon d t o Asp102. Th e oxya n ion of t h e
t et r a h edr a l in t er m edia t e is st a bilized by in t er a ct ion
wit h t h e m a in ch a in NH s of t h e oxya n ion h ole. Th e
t et r a h edr a l in t er m edia t e colla pses wit h expu lsion of
lea vin g gr ou p, a ssist ed by H is57-H + a ct in g a s a
gen er a l a cid, t o yield t h e a cylen zym e in t er m edia t e.
F ig u re 4. Ca t a lysis of pept ide h ydr olysis. Th e ca t a lysis
of pept ide h ydr olysis in volves a ct iva t ion of t h e ca r bon yl via
a gen er a l a cid, a ct iva t ion of wa t er wit h a gen er a l ba se, a n d
pr ot on a t ion of t h e a m in e lea vin g gr ou p.
4506 Chemical Reviews, 2002, Vol. 102, No. 12
Hedstrom
F ig u re 5. Th e gen er a lly a ccept ed m ech a n ism for ser in e pr ot ea ses.
Th e dea cyla t ion h a lf of t h e r ea ct ion essen t ia lly
r epea t s t h e a bove sequ en ce: wa t er a t t a cks t h e a cylen zym e, a ssist ed by H is57, yieldin g a secon d t et r a h edr a l in t er m edia t e. Th is in t er m edia t e colla pses,
expellin g Ser 195 a n d ca r boxylic a cid pr odu ct . Th e
a cylen zym e in t er m edia t e is well est a blish ed a s discu ssed below, bu t t h e t et r a h edr a l in t er m edia t es a r e
in fer r ed fr om solu t ion ch em ist r y. Th e t r a n sit ion
st a t es of t h e a cyla t ion a n d dea cyla t ion r ea ct ion s will
r esem ble t h e h igh en er gy t et r a h edr a l in t er m edia t es,
a n d t h e t er m s “t r a n sit ion st a t e” a n d “t et r a h edr a l
in t er m edia t e” a r e oft en u sed in discr im in a t ely in t h e
lit er a t u r e. It is wor t h n ot in g t h a t a web of h ydr ogen
bon din g in t er a ct ion s lin ks t h e su bst r a t e bin din g sit es
t o t h e ca t a lyt ic t r ia d. As t h e r ea ct ion pr oceeds,
ch a n ges in bon din g a n d ch a r ge a t t h e scissile bon d
will pr opa ga t e t o m or e r em ot e en zym e-su bst r a t e
in t er a ct ion s, a n d vice ver sa . Th e eviden ce su ppor t in g
t h is m ech a n ism h a s been r eviewed ext en sively, a n d
will be on ly br iefly descr ibed h er e.19,56,60-62
1. The Discovery of the Catalytic Triad and the Oxyanion
Hole
In it ia l in vest iga t ion s of ser in e pr ot ea ses focu sed on
en zym es t h a t cou ld be r ea dily obt a in ed in la r ge
qu a n t it ies: bovin e ch ym ot r ypsin a n d t r ypsin a n d
por cin e ela st a se. In a ct iva t or s r evea led t h e fir st clu es
in t o t h e ca t a lyt ic m a ch in er y. Ner ve ga ses su ch a s
diisopr opylflu or oph osph a t e (DF P ) in a ct iva t e ch ym ot r ypsin by specifica lly m odifyin g Ser 195 (F igu r e
6)63-65 a n d H is57 is m odified wh en ch ym ot r ypsin is
in a ct iva t ed by t osyl-L -ph en yla la n in e ch lor om et h yl
ket on e (TP CK).66 Th e in volvem en t of H is57 wa s a lso
su ggest ed by t h e pH r a t e pr ofiles wh ich in dica t ed
t h a t t h e depr ot on a t ion of a r esidu e wit h pK a ∼ 7 wa s
im por t a n t for a ct ivit y.56 Su bsequ en t NMR exper iments showed that the pK a of His57 is ∼7.21 The third
member of the catalytic triad, Asp102, was discovered
wh en Blow solved t h e cr yst a l st r u ct u r e of ch ym ot r ypsin .67 Blow pr oposed t h a t t h e h ydr ogen bon din g
n et wor k bet ween Asp102, H is57, a n d Ser 195st h e
ch a r ge r ela y syst em sa ct iva t es Ser 195 for n u cleoph ilic a t t a ck. A ch a r ge r ela y syst em wa s r a pidly
iden t ified in t r ypsin , ela st a se, a n d ot h er r ela t ed
en zym es,32,33,68 a n d a sim ila r ca t a lyt ic t r ia d of Asp32,
H is 64, a n d Ser 221 wa s discover ed in su bt ilisin .69,70
Th e oxya n ion h ole wa s fir st n ot ed by H en der son , wh o
pr oposed t h a t t h e m a in ch a in NH s of Gly193 a n d
Ser 195 st a bilize t h e n ega t ively ch a r ged oxya n ion of
F ig u re 6. Mech a n ism in h ibit or s t h a t for m t et r a h edr a l
a ddu ct s.
t h e t et r a h edr a l in t er m edia t e.71 Cr yst a l st r u ct u r es of
sever a l t r a n sit ion st a t e a n a logu e com plexes con fir m
t h is in t er a ct ion .33,45,72-74
2. Mutagenesis of the Catalytic Triad
Ma n y exper im en t s con fir m t h e im por t a n ce of t h e
ca t a lyt ic t r ia d r esidu es in ser in e pr ot ea se ca t a lysis.
Ch em ica l m odifica t ion exper im en t s wer e fir st u sed
t o disr u pt t h e ca t a lyt ic t r ia d, eit h er r em ovin g t h e
h ydr oxyl of Ser 195 t o cr ea t e a n h ydr o pr ot ea ses or
m et h yla t in g H is57 a t N!2.75,76 E it h er m odifica t ion
decr ea ses pr ot ea se a ct ivit y by a t lea st 10 4 -fold.77,78
Sit e-dir ect ed m u t a gen esis exper im en t s pr ovided a
m or e in cisive m ea n s t o a lt er t h e ca t a lyt ic t r ia d. In
t r ypsin , t h e su bst it u t ion of eit h er Ser 195 or H is57
wit h Ala decr ea ses t h e va lu e of k cat /K m by 10 5.79 (Su ch
va lu es sh ou ld m ost r igor ou sly be con sider ed u pper
lim it s for t h e a ct ivit y of a given m u t a n t en zym e.
Wa t er ca n su bst it u t e for a m issin g fu n ct ion a l gr ou p,
t h u s pr ovidin g a n a lt er n a t ive m ech a n ism of ca t a lysis
a n d u n der est im a t in g t h e effect of t h e m u t a t ion . In
addition, factors such as translation misincorporation
Serine Protease Mechanism and Specificity
F ig u re 7. Th e P 2 a n d P 1ʹ′ r esidu es br a cket H is57. Th e
P 2 a n d P 1ʹ′ r esidu es fr om t h e ch ym ot r ypsin -t u r key ovom u coid t h ir d dom a in com plex a t 2.0 Å, P DB a ccession
n u m ber 1CH O. Th is figu r e wa s pr odu ced wit h Molscr ipt
v2.1 (r ef 267) a n d P OV-Ra y by Lu Ga n .
a n d con t a m in a t in g en zym es m u st a lso be con sider ed
wh en eva lu a t in g m u t a n t en zym es wit h ver y low
a ct ivit ies.) No fu r t h er loss of a ct ivit y is obser ved
wh en t h e r em a in in g ca t a lyt ic t r ia d r esidu es a r e
su bst it u t ed. Th u s, t h e su bst it u t ion of eit h er H is57
or Ser 195 is su fficien t t o com plet ely disa ble t h e
ca t a lyt ic t r ia d. Th e su bst it u t ion of Asp102 t o Asn in
t r ypsin decr ea ses k ca t /K m by 10 4 a t n eu t r a l pH .80,81
Th e a n a logou s m u t a t ion s of t h e ca t a lyt ic t r ia d h a ve
similar effects in subtilisin.82 However, it is important
t o r ecogn ize t h a t t h e ca t a lyt ic t r ia d is n ot t h e sole
sou r ce of pr ot ea se ca t a lyt ic power : even wit h t h e
ca t a lyt ic t r ia d disa bled, t h ese m u t a n t pr ot ea ses
h ydr olyze su bst r a t es a t ∼10 3 × t h e u n ca t a lyzed
r a t e.79,82 Th is r em a in in g a ct ivit y r eflect s t h e con t r ibu t ion of t h e oxya n ion h ole, desolva t ion , a n d per h a ps
t h e ca n on ica l con for m a t ion t o ca t a lysis.
In t er est in gly, t h e a ct ivit y of H is57 m u t a t ion s ca n
be r escu ed by su bst r a t es con t a in in g H is. Th is su bst r a t e a ssist ed ca t a lysis wa s fir st dem on st r a t ed in
su bt ilisin a n d h a s r ecen t ly been ext en ded t o t r ypsin .83-86 In t h e a bsen ce of H is57, t h e pr esen ce of P 2H is or P 1ʹ′-H is in cr ea ses pept ide h ydr olysis by a s
m u ch a s 300-fold. As n ot ed a bove, H is57 in t er a ct s
wit h bot h t h e P 2 a n d P 1ʹ′ r esidu es (F igu r e 7); it is
believed t h a t t h e P 2- a n d P 1ʹ′-H is ca n r epla ce H is57
in t h e ca t a lyt ic t r ia d, for m in g sim ila r in t er a ct ion s
wit h Ser 195 a n d Asp102.
A secon d in st a n ce of ch em ica l r escu e is fou n d in
m u t a t ion s of Asp102. H ydr oxide ion in cr ea ses t h e
a ct ivit y of Asp102Asn t r ypsin t o wit h in 10% of wild
t ype a t pH 10.80 Sim ila r h ydr oxide a ct iva t ion is a lso
obser ved in su bt ilisin .82 Th e m ech a n ism of t h is
h ydr oxide r escu e is n ot u n der st ood.
Un for t u n a t ely, sin ce t h e oxya n ion h ole of ch ym ot r ypsin -like en zym es is com posed of m a in ch a in a t om s, sit e dir ect ed m u t a gen esis ca n n ot be u sed t o
con fir m it s r ole in ca t a lysis. Th e oxya n ion h ole of
su bt ilisin is com posed of t h e side ch a in s of Asn 155
a n d Th r 220 a s well a s t h e ba ckbon e NH of Ser 221.
Su bst it u t ion of Asn 155 decr ea ses k cat /K m by 100-10 3fold, in dica t in g t h a t t h is r esidu e pr ovides 3-4 kca l/
m ol of t r a n sit ion st a t e st a biliza t ion en er gy. 87-90 Th e
su bst it u t ion of Th r 220 wit h Ala decr ea ses k ca t /K m by
∼20-fold (∼2 kca l/m ol) a n d t h e r em ova l of bot h
Asn 155 a n d Th r 220 decr ea ses a ct ivit y by 10 4 -fold,
su ggest in g t h a t t h e oxya n ion h ole ca n st a bilize t h e
t r a n sit ion st a t e by a s m u ch a s 6 kca l/m ol. H owever ,
n o fu r t h er loss of a ct ivit y is obser ved wh en oxya n ion
Chemical Reviews, 2002, Vol. 102, No. 12 4507
m u t a t ion s a r e com bin ed wit h m u t a t ion s of t h e ca t a lyt ic t r ia d, in dica t in g t h a t t h e oxya n ion a n d ca t a lyt ic
t r ia d fu n ct ion cooper a t ively. Th er efor e, t h e 6 kca l/
m ol of t r a n sit ion st a t e st a biliza t ion is n ot sim ply t h e
m ea su r e of t h e in t er a ct ion s a t t h e oxya n ion h ole.90
Th e colla bor a t ive fu n ct ion of t h e ca t a lyt ic m a ch in er y
m a kes it im possible t o pr ecisely a scr ibe en er gies t o
in dividu a l in t er a ct ion s. In t er est in gly, Th r 220 a ppea r s t o be t oo fa r a wa y t o h ydr ogen bon d dir ect ly t o
a t et r a h edr a l in t er m edia t e; eit h er Th r 220 m oves
du r in g ca t a lysis, or st a biliza t ion r esu lt s fr om lon gr a n ge elect r ost a t ic in t er a ct ion s. Molecu la r m odelin g
exper im en t s su ppor t bot h m ech a n ism s.88,91
3. Evidence for the Acylenzyme Intermediate
Th e fa m ou s bu r st exper im en t of H a r t ley a n d Kilbey wa s t h e fir st eviden ce for t h e a cylen zym e in t er m edia t e.92 Th e h ydr olysis of p-n it r oph en yl a cet a t e by
ch ym ot r ypsin is biph a sic, wit h on e en zym e-equ iva len t of p-n it r oph en ol pr odu ced in t h e r a pid ph a se,
in dica t in g t h a t a n en zym e-a cet a t e in t er m edia t e
for m ed a n d decom posit ion of t h is in t er m edia t e wa s
r a t e-lim it in g. Acylen zym e in t er m edia t es wer e su bsequ en t ly isola t ed in sever a l r ea ct ion s of est er s wit h
ch ym ot r ypsin , a n d t h e ch em ica l/kin et ic com pet en ce
of these intermedia tes wa s demonstra ted.56 However,
sim ila r exper im en t s fa iled t o det ect a cylen zym e
in t er m edia t es in t h e h ydr olysis of a m ide su bst r a t es.
Th is fa ilu r e cou ld be r ea dily expla in ed if t h e for m a t ion of t h e a cylen zym e wa s r a t e-lim it in g, so t h a t t h e
a cylen zym e n o lon ger a ccu m u la t ed t o det ect a ble
levels du r in g a m ide h ydr olysis. Th e a cylen zym e
intermediate could be detected in acyltransfer experim en t s.93 As sh own in F igu r e 8, t h e a cylen zym e
F ig u re 8. Th e m ech a n ism of a cylt r a n sfer r ea ct ion s of
ser in e pr ot ea ses. Th e a cylen zym e n or m a lly r ea ct s wit h
wa t er t o for m a ca r boxylic a cid. H owever , ot h er n u cleoph iles ca n a lso r ea ct wit h t h e a cyl en zym e. If t h e a dded
n u cleoph ile is t h e N-t er m in u s of a pept ide, t h e r ea ct ion is
the reverse of the hydrolysis reaction and displays the same
specificit y.
u su a lly r ea ct s wit h wa t er , bu t ca n a lso r ea ct wit h
ot h er n u cleoph iles. Th e pa r t it ion in g of a cylt r a n sfer
bet ween wa t er a n d n u cleoph ile will be con st a n t for
a given a cylen zym e, r ega r dless of h ow t h e a cylen zym e wa s for m ed. E st er s a n d a m ides h a ve iden t ical partition ratios in acyltransfer reaction, indicating
t h a t t h eir r ea ct ion s in volves a com m on a cylen zym e.
Not e t h a t if t h e n u cleoph ile is a pept ide, t h e a cylt r a n sfer r ea ct ion is t h e r ever se of pept ide h ydr olysis,
a n d m u st exh ibit t h e sa m e specificit y. Th ese r ea ct ion s h a ve pr oven u sefu l in m a ppin g t h e specificit y
of t h e Sʹ′ sit es.94-99
Sever a l X-r a y cr yst a l st r u ct u r es of a cylen zym e
in t er m edia t es h a ve been solved. Th e fir st st r u ct u r e
of a pept idyl a cylen zym e wa s a su r pr ise r evea led
wh en h igh -r esolu t ion da t a u n cover ed a pept ide in t h e
a ct ive sit es of γ-ch ym ot r ypsin .41,100,101 Th e pept ide is
4508 Chemical Reviews, 2002, Vol. 102, No. 12
pr oba bly P r o-Gly-Ala -Tyr , bu t m a y be a m ixt u r e
der ived fr om a u t opr ot eolysis. At low pH , con t in u ou s
den sit y is obser ved bet ween Ser 195 a n d t h e ca r bon yl
of t h e pept ide, in dica t in g t h a t t h e a cylen zym e is
for m ed. In t er est in gly, t h e ca r bon yl gr ou p is n ot
com plet ely in t h e oxya n ion h ole: t h e ca r bon yl oxygen
for m s a h ydr ogen bon d wit h Gly193 (2.8 Å), bu t n ot
wit h Ser 195 (3.5 Å). At h igh pH , t h e cova len t bon d
bet ween Ser 195 a n d t h e pept ide is lost , in dica t in g
t h a t t h e pr odu ct com plex h a s for m ed. Th e pept ide
ca n be r ea dily r epla ced wit h in h ibit or s, fu r t h er
dem on st r a t in g t h a t t h is a cylen zym e is ca t a lyt ica lly
com pet en t . A pept ide wa s a lso discover ed in t h e
a ct ive sit e of S treptom yces griseu s pr ot ein a se A
(SGP A).42 Th is pept ide a ppea r s t o be a m ixt u r e of
a u t olysis pr odu ct s. As in γ-ch ym ot r ypsin , t h e pept ide
a ppea r s t o be a n a cylen zym e a t low pH a n d a pr odu ct
com plex a t h igh pH . Un like γ-ch ym ot r ypsin , t h e
ca r bon yl oxygen of t h e a cylen zym e occu pies t h e
oxya n ion h ole. Mor e r ecen t ly, Wilm ou t h a n d collea gu es scr een ed a pept ide libr a r y t o isola t e a st a ble
pept idyl a cylen zym e of ela st a se.102 β-Ca som or ph in is
a h ept a pept ide t h a t for m s a st a ble a cylen zym e a t low
pH via t h e C-t er m in a l ca r boxyla t e. Th e ca r bon yl
oxygen is a lso fou n d in t h e oxya n ion h ole in t h is
a cylen zym e. Alt h ou gh in it ia l r epor t s su ggest ed t h a t
t h is ca r bon yl gr ou p wa s dist or t ed t owa r d a t et r a h edr a l st r u ct u r e, m or e r ecen t h igh -r esolu t ion da t a
in dica t e t h a t t h e est er is pla n a r .102 Th e st a bilit y of
t h ese a cylen zym es ca n be a t t r ibu t ed t o t h e pr ot on a t ion of H is57 a t low pH . All t h r ee of t h ese pept idyl
a cylen zym es displa y t h e ca n on ica l con for m a t ion .
4. Evidence for the Tetrahedral Intermediate
As yet , t h e t et r a h edr a l in t er m edia t e h a s n ot been
r igor ou sly dem on st r a t ed in t h e ser in e pr ot ea se r ea ct ion , bu t is in clu ded on t h e ba sis of solu t ion ch em ist r y.103,104 Tet r a h edr a l in t er m edia t es a r e u n st a ble
a n d h a ve ver y sh or t lifet im es.78 F u r t h er , t h e lifet im e
of t h e in t er m edia t e decr ea ses a s t h e lea vin g gr ou p
im pr oves. In fa vor a ble cir cu m st a n ces, su ch lea vin g
gr ou p effect s ca n pr ovide eviden ce for t et r a h edr a l
in t er m edia t es: a s t h e pK a of t h e lea vin g gr ou p
decr ea ses below t h a t of t h e n u cleoph ile, for m a t ion of
t h e t et r a h edr a l in t er m edia t e becom es r a t e-lim it in g
a n d a ch a r a ct er ist ic br ea k will be obser ved in t h e
Br on st ed plot .105 In t h e ca se of t h e best lea vin g
gr ou ps su ch a s p-n it r oph en ol, t h e lifet im e of t h e
t et r a h edr a l in t er m edia t e becom es less t h a n a vibr a t ion , t h e r ea ct ion becom es con cer t ed a n d t h e t et r a h edr a l species m u st be con sider ed a t r a n sit ion
st a t e.106-108 As in solu t ion , t h e h ydr olysis of pn it r oph en yla cet a t e by ch ym ot r ypsin is a con cer t ed
r ea ct ion ,109 a n d it is for m a lly possible t h a t en zym eca t a lyzed pept ide h ydr olysis is a lso con cer t ed. Of
cou r se, t h is wou ld r equ ir e a n en or m ou s a ct iva t ion of
t h e a m in e lea vin g gr ou p. Un for t u n a t ely, t h e lea vin g
gr ou p effect s m ea su r ed in Br on st ed a n a lysis a r e
gr ea t ly r edu ced by gen er a l ba se ca t a lysis, so t h a t
Br on st ed a n a lysis of ch ym ot r ypsin ca t a lyzed r ea ct ion s pr ovides n o in for m a t ion a bou t t h e t et r a h edr a l
in t er m edia t e.110,111 14 N/15 N isot ope effect s h a ve been
sim ila r ly equ ivoca l.62
Hedstrom
Not su r pr isin gly, t h e obser va t ion of t h e t et r a h edr a l
in t er m edia t e by X-r a y cr yst a llogr a ph y h a s pr oven
elusive.112 The first report of a tetrahedral intermedia t e in t h e t r ypsin -BP TI com plex cou ld n ot be su bst a n t ia t ed by NMR exper im en t s; fu r t h er r efin em en t
r evea led t h a t t h e scissile pept ide bon d is dist or t ed
t owa r d t et r a h edr a l, bu t n o cova len t bon d is for m ed
wit h Ser 195.113,114 In t er est in gly, t h is bon d is a lso
dist or t ed in t h e a bsen ce of t r ypsin . 115 An ot h er t et r a h edr a l in t er m edia t e h a s r ecen t ly been r epor t ed in
cr yst a ls of ela st a se a n d t h e pept ide β-ca som or ph in .102
Wh en cr yst a ls con t a in in g t h e pept idyl a cylen zym e
a r e t r a n sfer r ed t o pH 9.0 a n d fla sh -fr ozen , elect r on
den sit y is obser ved t h a t su ggest s a t et r a h edr a l a ddu ct . H owever , t h is excit in g r esu lt m u st be a ppr oa ch ed wit h gr ea t ca u t ion . X-r a y cr yst a llogr a ph y
is a t im e a ver a ged m et h od, a n d t h is t et r a h edr a l
a ddu ct m a y be a m ixt u r e of com plexes. Th e pr esen ce
of su lfa t e in t h e cr yopr ot ect a n t solu t ion is especia lly
t r ou blin g beca u se su lfa t e bin ds in t h e a ct ive sit e of
m a n y ser in e pr ot ea ses a n d ca n ea sily be m ist a ken
for a t et r a h edr a l in t er em edia t e (see r efs 116 a n d 117
a n d r efer en ces t h er ein ). Th e kin et ics of in t er m edia t e
for m a t ion a r e a lso n ot con sist en t wit h a pseu do fir st
order process (the tetrahedral intermediate is present
a t 1 m in bu t com plet ely decom posed a ft er 2 m in ),
wh ich fu r t h er su ggest s t h a t t h e “t et r a h edr a l in t er m edia t e” is a ct u a lly a m ixt u r e of com plexes. A m or e
pr om isin g t et r a h edr a l in t er m edia t e a ppea r s wh en
cr yst a ls t h e pept idyl a cylen zym e of γ-ch ym ot r ypsin
a r e soa ked in h exa n e.118 Th e elect r on den sit y a r ou n d
t h e a cyl ca r bon yl a ppea r s t o be t et r a h edr a l, a n d
density is observed in the P1ʹ′ site. These observations
su ggest t h a t a pept ide fr om t h e cr yst a lliza t ion m ix
h a s a t t a cked t h e a cylen zym e, for m in g a st a ble t et r a h edr a l in t er m edia t e. Th e st a bilit y of t h is in t er m edia t e is n ot u n der st ood, bu t m igh t be expla in ed by a
sh ift in equ ilibr iu m a n d/or h ydr ogen bon din g en er gies in or ga n ic solven t . Un for t u n a t ely, t et r a h edr a l
in t er m edia t es h a ve com e a n d gon e; t h e pu t a t ive
t et r a h edr a l in t er m edia t es of bot h ela st a se a n d ch ym ot r ypsin sh ou ld be su bst a n t ia t ed by a ddit ion a l
m et h ods.
B. Mechanisms of Inactivation and Inhibition
A com pr eh en sive discu ssion of in h ibit or /in a ct iva t or
m ech a n ism s ca n be fou n d in t h e P ower s pa per in t h is
issu e. Th e followin g will be a br ief discu ssion of
com pou n ds t h a t h a ve pla yed a n im por t a n t r ole in
u n r a velin g t h e m ech a n ism of ser in e pr ot ea se ca t a lysis a n d su bst r a t e specificit y.
1. Transition State Analogue Inhibitors
Several classes of inhibitors form stable tetrahedral
a ddu ct s t h a t m im ic t h e t et r a h edr a l in t er m edia t e/
t r a n sit ion st a t e of t h e ser in e pr ot ea se r ea ct ion . Th e
specificit y of t h ese r ea gen t s ca n be t a ilor ed t o a given
pr ot ea se by com bin in g t h e r ea ct ive gr ou ps wit h t h e
a ppr opr ia t e pept idyl por t ion s.72,119 DF P a n d t h e less
t oxic ph en ylm et h a n esu lfon ylflu or ide (P MSF ) in a ct iva t e vir t u a lly a ll ser in e pr ot ea ses a n d a r e dia gn ost ic
for t h is pr ot ea se cla ss. Th ese com pou n ds a r e ver y
elect r oph ilic, a n d r ea ct ir r ever sibly wit h Ser 195 t o
for m st a ble t et r a h edr a l a ddu ct s (F igu r e 6). Ch lor o-
Serine Protease Mechanism and Specificity
F ig u re 9. Mech a n ism s of in h ibit or s t h a t for m st a ble a cylen zym es.
m et h yl ket on es (CMK) a r e pot en t in a ct iva t or s of bot h
ser in e a n d cyst ein e pr ot ea ses, a n d t h er efor e a r e n ot
dia gn ost ic.120 Ch lor om et h yl ket on es cr oss-lin k ser in e
pr ot ea ses by for m in g a h em iket a l wit h Ser 195 a n d
a lkyla t in g H is57;121-123 t h e r ea ct ion pr oceeds via a n
epoxide in t er m edia t e a s sh own in F igu r e 6. 124-127
Interestingly, the formation of the epoxide intermedia t e r equ ir es t h a t t h e oxya n ion ca n esca pe t h e oxya n ion h ole.
P ept idyl a ldeh ydes, t r iflu or om et h yl ket on es, bor on ic a cids, a n d sim ila r com pou n ds for m r ever sible
t et r a h edr a l a ddu ct s wit h Ser 195. Th ese com plexes
closely r esem ble t h e t r a n sit ion st a t e/t et r a h edr a l in t er m edia t e of t h e h ydr olysis r ea ct ion : H is57 is pr ot on a t ed a n d t h e h em iket a l oxygen is n ega t ively
ch a r ged a n d bou n d in t h e oxya n ion h ole (F igu r e 6).
Th e a ffin it y of t h ese in h ibit or s in cr ea ses a s t h ey
in cor por a t e t h e fea t u r es of good su bst r a t es; a ffin it y
cor r ela t es wit h t h e va lu e of k ca t /K m for t h e h ydr olysis
of t h e a n a logou s su bst r a t e, a s r equ ir ed for a t r a n sit ion st a t e a n a logu e.47,49,128 Som e a ldeh yde in h ibit or s
a lso form a n a lter n a t ive a ddu ct wh er e t h e h em ia ceta l
oxygen interacts with His57 rather than the oxyanion
h ole.129-131 Beca u se t h is a ddu ct m im ics t h e in t er a ct ion s of t h e lea vin g gr ou p, it ca n a lso be con sider ed
a t r a n sit ion st a t e a n a logu e. H owever , pept idyl bor on ic a cids ca n a lso for m a n a ddu ct a t N!2 of H is57
that cannot be considered a tra nsition sta te analog.132
Th ese Type 2 a ddu ct s a r e for m ed wh en in h ibit or s
r esem ble poor su bst r a t es or in com plexes of pr ot ea se
zym ogen s,133 for m in g h igh er a ffin it y com plexes t h a n
wou ld be expect ed ba sed on t h e h ydr olysis of a n a logou s su bst r a t es.
2. Stable Acylenzymes
Ma n y com pou n ds in a ct iva t e ser in e pr ot ea ses by
for m in g st a ble a cylen zym e in t er m edia t es. Th is st a bilit y der ives fr om t h r ee gen er a l m ech a n ism s:
(a ) Th e in t r in sic r ea ct ivit y of t h e a cyl gr ou p. Th e
r ea ct ivit y of est er s ca n be decr ea sed by in cr ea sin g
t h e elect r on den sit y of t h e ca r bon yl. F or exa m ple, t h e
dea cyla t ion r a t es of ben zoyl ch ym ot r ypsin s decr ea se
as the substituent becomes more electron-donating.134
Sim ila r ly, a lph a h et er oa t om s a lso dea ct iva t e est er s,
a n d a cylch ym ot r ypsin s der ived fr om a za pept ides a r e
st a ble.135 Not e t h a t a za pept idyl a cylen zym es a r e
isost er ic wit h pept idyl a cylen zym es, a n d t h u s a r e
Chemical Reviews, 2002, Vol. 102, No. 12 4509
expect ed t o closely m im ic t h e n or m a l ca t a lyt ic in t er m edia t es.136 Of cou r se, t h ese effect s a lso decr ea se t h e
a cyla t ion r a t e con st a n t s, m a kin g it m or e difficu lt t o
for m t h e a cylen zym e. H owever , t h e effect s on a cyla t ion ca n be offset wit h good lea vin g gr ou ps su ch a s
p-n it r oph en ol. Not e t h a t su ch r ea gen t s a s p-n it r oph en yl-gu a n idin oben zoa t e a r e a ct ive sit e t it r a n t s,
r ea ct in g wit h t r ypsin t o for m a st a ble a cylen zym e
in t er m edia t e a n d on e equ iva len t of p-n it r oph en ol
(F igu r e 9).137 E lect r on -wit h dr a win g pr oper t ies ca n
a lso be disgu ised by m or e clever m ea n s a n d m a n y
cla sses of m ech a n ism -ba sed in a ct iva t or s exploit t h is
st r a t egy (see P ower s pa per in t h is issu e).
(b) Th e a cylen zym e does n ot in t er a ct wit h t h e
oxya n ion h ole. Th is m ech a n ism is illu st r a t ed by
a r yla cr yloyl ch ym ot r ypsin , wh er e t h e pla n a r it y of t h e
a r yla cr yloyl syst em does n ot a llow t h e ca r bon yl t o
enter the oxyanion hole.71,138-141 A similar mechanism
m a y a ccou n t for t h e st a bilit y of a cet yl-ch ym ot r ypsin ;
t h is a ddu ct la cks a la r ge h ydr oph obic gr ou p t h a t ca n
bin d in t h e S1 sit e a n d dir ect t h e ca r bon yl oxygen
in t o t h e oxya n ion h ole.
(c) Th e ca t a lyt ic t r ia d is disr u pt ed a n d ca n n ot
a ct iva t e wa t er . Th e sim plest m a n ifest a t ion of t h is
m ech a n ism is t h e pr ot on a t ion of H is57 so t h a t it
ca n n ot ser ve a s a gen er a l ba se ca t a lyst a s discu ssed
a bove. A secon d exa m ple is t h e in a ct iva t ion of ch ymotrypsin by 3-benzyl-6-chloro-2-pyrone.142 This compou n d for m s a st a ble a cylen zym e wh er e Ser 195
attaches to C1 of (Z)-2-benzylpentenedioic acid. His57
for m s a sa lt br idge wit h t h e fr ee ca r boxyla t e of t h e
in a ct iva t or .143 Th is sa lt br idge pr even t s H is57 fr om
per for m in g it s r ole a s t h e gen er a l ba se in t h e dea cyla t ion r ea ct ion . Na t u r e h a s a lso u sed t h is st r a t egy
t o in h ibit ser in e pr ot ea ses. Ser pin s for m st a ble
a cylen zym es; t h is st a bilit y r esu lt s fr om dist or t ion of
t h e a ct ive sit e u pon com plex for m a t ion 144 (see t h e
Get t in s pa per in t h is issu e).
C. Issues in the Function of the Catalytic Triad
1. Where Are the Protons? One-Proton versus
Two-Proton-Transfer Mechanisms
Alt h ou gh t h e gen er a l fea t u r es of t h e ch a r ge r ela y
syst em a r e widely a ccept ed, t h e det a ils of it s fu n ct ion
h a ve been su r pr isin gly con t r over sia l.21 Th e issu e of
wh et h er t h e pr ot on in t h e Asp102-H is57 h ydr ogen
bon d r esides on H is57 or Asp102 h a s been pa r t icu la r ly con t en t iou s. Blow’s or igin a l con cept ion of t h e
ch a r ge r ela y syst em expla in ed t h e r ea ct ivit y of
Ser 195 in t er m s of r eson a n ce for m s Asp-CO 2 -/H -H is/
Ser -OH a n d Asp-CO 2 H /H is-H /Ser -O -.1,67 Th is m ech a n ism wa s la t er r efin ed t o in volve a ct u a l pr ot on
t r a n sfer s fr om Ser 195 t o H is57 a n d H is57 t o Asp102,
r esu lt in g in t h e t et r a h edr a l in t er m edia t e a n d n eu t r a l
Asp102 a n d H is57.145,146 Th is t wo-pr ot on -t r a n sfer
mechanism was controversial from the outset because
it r equ ir es t h a t t h e pK a of H is57 is low er t h a n
Asp102, i.e., t h e pK a of H is57 m u st decr ea se in t h e
t et r a h edr a l in t er m edia t e/t r a n sit ion st a t e. Never t h eless, t h is m ech a n ism wa s in cor por a t ed in t o m a n y
t ext books.
1 H NMR exper im en t s st r on gly fa vor t h e on e-pr ot on
m ech a n ism . Th e Nδ1 pr ot on of H is57 h a s a n u n u su -
4510 Chemical Reviews, 2002, Vol. 102, No. 12
a lly low field 1 H NMR sign a l (13.8-15 a n d 17-18
ppm in n eu t r a l a n d pr ot on a t ed H is57, r espect ively)
wh ich ca n be u sed t o det er m in e t h e pK a of H is57 in
va r iou s com plexes.147,148 Th e pK a of H is57 is ∼7 in
t h e fr ee en zym e, a cylen zym e, a n d pr ot ein in h ibit or
com plexes bu t in creases t o gr ea t er t h a n 10 in t r a n sit ion st a t e a n a logu e com plexes.128,149-151 Neu t r on diffr a ct ion exper im en t s a lso dem on st r a t ed t h a t t h e
pr ot on is a ssocia t ed wit h H is57 in m on oisopr opyl
ph osph a t e m odified t r ypsin .152 Alt h ou gh t h ese exper im en t s do n ot est a blish t h e posit ion of t h e pr ot on
in t h e t r a n sit ion st a t e of t h e ca t a lyt ic r ea ct ion , t h ey
a r gu e st r on gly for t h e on e-pr ot on m ech a n ism , wh er e
pr ot on a t ed H is57 is st a bilized by a n elect r ost a t ic
in t er a ct ion wit h a n ega t ively ch a r ged Asp102.
2. Low Barrier Hydrogen Bonds
A m or e su bt le ver sion of t h e t wo-pr ot on -t r a n sfer
m ech a n ism h a s em er ged in t h e low ba r r ier h ydr ogen
bon d (LBH B, a lso kn own a s sh or t , st r on g h ydr ogen
bon d) h ypot h esis of r ecen t yea r s.153-159 Th is con t r over sia l t h eor y developed fr om t h e obser va t ion t h a t
u n u su a l h ydr ogen bon ds for m in t h e ga s ph a se,
ch a r a ct er ized by sh or t len gt h (<2.6 Å) a n d ext r a or din a r y st r en gt h (30 kca l/m ol).160,161 Th e u n u su a l
pr oper t ies of t h ese h ydr ogen bon ds is believed t o
der ive fr om t h e sh or t dist a n ce bet ween don or a n d
a ccept or a t om s wit h m a t ch ed pK a ’s, wh ich elim in a t es
t h e ba r r ier for pr ot on t r a n sfer so t h a t t h e pr ot on is
sh a r ed in a br oa d sin gle-pot en t ia l well. Th e h ydr ogen
bon d bet ween Asp102 a n d H is57 a ppea r s t o fu lfill
m a n y of t h e cr it er ia for a LBH B:162-164 (a ) Th e
dist a n ce bet ween Asp102 a n d H is57 is <2.6 Å. (b)
Th e 1 H NMR sign a l of t h e Nδ1 pr ot on of H is57 is
u n u su a lly down field.21,147 (c) A deu t er iu m isot ope
effect is obser ved on ch em ica l sh ift of t h e H is57 Nδ1
pr ot on . (d) Th e D/H fr a ct ion a t ion fa ct or of t h e Nδ1
pr ot on is low.150,165 Un for t u n a t ely, t h ese obser va t ion s
a r e n ot dia gn ost ic for LBH Bs, a n d t h e Asp-H is
h ydr ogen bon d fa ils by ot h er m ea su r es: (a ) Th e pK a ’s
of Asp102 and His57 a re not ma tched. (b) both 15N-H
spin cou plin gs a n d 15 N ch em ica l sh ift s in dica t e t h a t
t h e pr ot on is ∼85% loca lized on H is57.158,166 (c) Th e
H is57-Nδ1 pr ot on is n ot sh ielded fr om wa t er a n d
t h e en vir on m en t is pola r .158 Mor eover , bot h Asp102
a n d H is57 a r e in volved in m u lt iple h ydr ogen bon din g
interactions; these intera ctions will compete with a nd
wea ken t h e Oδ1-Nδ1-H h ydr ogen bon d. F u r t h er ,
disr u pt ion of t h e ca t a lyt ic t r ia d decr ea ses t h e ca t a lyt ic power of ser in e pr ot ea ses by n o m or e t h a n ∼6
kca l/m ol, wh ich m u st be dist r ibu t ed a m on g sever a l
h ydr ogen bon ds in a ddit ion t o t h e Asp102-H is57
in t er a ct ion . Th is en er gy loss is con sist en t wit h a
sim ple elect r ost a t ic in t er a ct ion .167 La st , m u t a t ion s a t
Asp102 ca n be r escu ed by r epla cin g Ser 214 wit h
Asp.168 Alt h ou gh t h is a lt er ed ca t a lyt ic t r ia d h a s n ot
been thoroughly cha ra cterized, it would be surprisin g
if a LBH B cou ld be so ea sily r econ st it u t ed. Sim ila r ly,
su bst it u t ion of t h e ca t a lyt ic t r ia d Asp32 wit h Cys in
su bt ilisin elim in a t es t h e pu t a t ive LBH B, bu t decr ea ses k ca t /K m by n o m or e t h a n 50-fold.169 Th ese
exper im en t s su ggest t h a t if a LBH B exist s, it con t r ibu t es n o m or e t h a n 2 kca l/m ol t o t h e ca t a lyt ic
power of ser in e pr ot ea ses.
Hedstrom
Th e in t er a ct ion s of t h e oxya n ion wit h t h e oxya n ion
h ole h a ve a lso been su ggest ed t o in volve LBH Bs. 153
H owever , t h e t wo h ydr ogen bon ds of t h e oxya n ion
h ole pr ovide a t ot a l of ∼6.5 kca l/m ol in t r a n sit ion
st a t e st a biliza t ion ,87,89,90,149,170 wh ich is con sist en t
wit h sim ple elect r ost a t ic in t er a ct ion s.171
3. Moving His Mechanisms
In t h e gen er a lly a ccept ed m ech a n ism for ser in e
pr ot ea se ca t a lysis (F igu r e 5), H is57 r em oves a pr ot on
fr om Ser 195 a n d t r a n sfer s it t o t h e lea vin g gr ou p.
Th is pr ot on t r a n sfer h a s t r ou bled som e in vest iga t or s,
wh o a r gu e t h a t if H is57 a bst r a ct s a pr ot on fr om
Ser195, then His57-H + will be positioned near Ser195
in t h e t et r a h edr a l in t er m edia t e, wh ich wou ld fa vor
r e-pr ot on a t ion of Ser 195 a n d r egen er a t ion of su bst r a t e.172,173 F or t h e r ea ct ion t o pr oceed, H is57-H +
m u st be posit ion ed t o pr ot on a t e t h e lea vin g gr ou p.
Th e “H is flip” m ech a n ism of F igu r e 10 h a s been
F ig u re 10. Th e “H is flip” m ech a n ism . Aft er in it ia l for m a t ion of t h e t et r a h edr a l in t er m edia t e, t h e N!1-H will be
wit h in h ydr ogen bon din g dist a n ce of t h e γO of Ser 195.
Rot a t ion of H is57 su pposedly pla ces t h e N!1-H n ea r t h e
lea vin g gr ou p NH . Alt h ou gh t h e h ydr ogen bon ds bet ween
Nδ1-H a n d Asp102 a n d C!1 a n d Ser 214 wou ld be disr u pt ed, n ew h ydr ogen bon ds will for m t o r epla ce t h em .21
pr oposed t o solve t h is pr oblem :27 a ft er for m a t ion of
t h e t et r a h edr a l in t er m edia t e, H is57-H + flips, wh ich
sh ou ld pla ce t h e Nδ1 pr ot on n ea r t h e lea vin g gr ou p.
Th e flipped con for m a t ion of H is57 h a s been obser ved
in su bt ilisin in 50% dim et h ylfor m a m ide. 174 In a ddit ion , t h e pr esen ce of t h e Ser 214-H is57 h ydr ogen
bon d a n d t h e u n u su a l dyn a m ics of H is57 h a ve been
t a ken a s eviden ce for t h e “H is flip” m ech a n ism .21
Sever a l con sider a t ion s a r gu e a ga in st t h e “H is flip”
m ech a n ism . F ir st , t h e disr u pt ion a n d r efor m a t ion of
so m a n y h ydr ogen bon ds in t h e sh or t lifet im e of t h e
tetra h edra l in term edia te seem s u n likely. The His flip
m ech a n ism a ppea r s t o viola t e t h e pr in ciple of lea st
m ot ion , wh ich su ggest s t h a t en zym a t ic r ea ct ion s
occu r wit h a n econ om y of m ovem en t . 175 Mor eover ,
H is57 is br a cket ed by t h e P 2 a n d P 1ʹ′ r esidu es of a
pept ide su bst r a t e, wh ich cr ea t es a st er ic ba r r ier t o
flippin g (F igu r e 7). Th e u n u su a l dyn a m ics of H is57
ca n be expla in ed by su bst r a t e a n d solven t m ovin g in
a n d ou t of t h e a ct ive sit e, or even t h e ch em ica l
t r a n sfor m a t ion st h er e is n o n eed t o in voke flippin g.
Most ser iou sly, wh ile t h e flippin g m ech a n ism looks
reasonable in two dimensions, it fails in three dimension s. An a lysis of pept ide t r iflu or oket on e or bor on ic
a cid com plexes sh ows t h a t t h e flip a ct u a lly pla ces
pr ot on don or fa r t h er a wa y fr om t h e lea vin g gr ou p by
0.8 Å.
Of cou r se, som e m ovem en t of bot h pr ot ea se a n d
su bst r a t e is in evit a ble a s t h e h ydr olysis r ea ct ion
pr oceeds. Ser 195 m u st m ove a t lea st 1 Å t o for m t h e
t et r a h edr a l in t er m edia t e a n d sm a ller m ovem en t s of
Serine Protease Mechanism and Specificity
t h e su bst r a t e will be r equ ir ed a s t h e bon din g ch a n ges
fr om sp 2 t o sp 3 . Th er efor e, H is57 m a y n ot be or ien t ed
t owa r d Ser 195 in t h e t et r a h edr a l in t er m edia t e a s
a ssu m ed. E ven if H is57 is posit ion ed so t h a t Ser 195
is pr ot on a t ed in t h e t et r a h edr a l in t er m edia t e, t h e
r ea ct ion is n ot doom ed t o colla pse ba ck t o st a r t in g
m a t er ia ls. Th e pr ot on is less t h a n 2.5 Å fr om t h e
a m in e lea vin g gr ou p, wh ich h a s t h e h igh er pK a .
P r ot on a t ion of t h e a m in e is fa vor ed over a ll, a n d t h e
pr ot on n eeds on ly t o t r a n sfer t o t h e a m in e befor e t h e
t et r a h edr a l in t er m edia t e br ea ks down . In a ddit ion ,
sever a l st r u ct u r es of t r a n sit ion st a t e a n a logu e com plexes in dica t e t h a t H is57-H + ca n in t er a ct wit h t h e
leaving group position while maintaining the Asp102H is57 h ydr ogen bon d (r ef 74 a n d r efer en ces t h er ein ).
Wh a t ever r eor ien t a t ion t h a t m a y be n ecessa r y for
com plet ion of t h e ca t a lyt ic cycle ca n pr oba bly be
a ccom plish ed wit h sm a ll a dju st m en t s in bot h pr ot ea se a n d su bst r a t e a n d wit h ou t br ea k in g t h e
Asp102-H is57 h ydr ogen bon d.
4. The Hydrolytic Water
Th e m ech a n ism of ser in e pr ot ea se ca t a lysis pla ces
som e r a t h er r igor ou s con st r a in t s on t h e h ydr olyt ic
wa t er : it sh ou ld a ppr oa ch a cylen zym e fr om t h e
lea vin g gr ou p side of t h e a ct ive sit e, it m u st m a ke a
h ydr ogen bon d t o H is57 a n d t h e a n gle of a t t a ck m u st
a ppr oxim a t e 109 degr ees. Sever a l cr yst a l st r u ct u r es
of ser in e pr ot ea ses con t a in or der ed wa t er m olecu les
t h a t a ppea r t o fu lfill on e or m or e of t h ese cr it er ia .
H en der son n ot ed t h e fir st su ch wa t er in a cin n a m oyl
en zym e com plex of ch ym ot r ypsin , a n d sim ila r ly
loca t ed wa t er s h a ve a ppea r ed in su bsequ en t a cylen zym e st r u ct u r es.41,71 Th is wa t er is 2.4 Å a bove t h e
ca r bon yl of t h e a cylen zym e a n d a lso in t er a ct s wit h
H is57 a s wou ld be expect ed of t h e h ydr olyt ic wa t er ,
bu t t h e a n gle of a t t a ck on t h e ca r bon yl is n ot
fa vor a ble. In a ddit ion , a sim ila r ly pla ced wa t er
m olecu le is obser ved in pr odu ct a n d t r a n sit ion st a t e
a n a logu e com plexes.176 Th e la t t er com plexes m im ic
t h e t et r a h edr a l in t er m edia t e, i.e., t h e in t er m edia t e
for m ed after t h e wa t er h a s a t t a cked; t h er efor e, t h is
wa t er is m or e likely t o pla y a st r u ct u r a l r ole. An ot h er
ca n dida t e wa t er m olecu le is obser ved in a ben zoylen zym e com plex of t r ypsin , poised 3.9 Å a bove t h e
ca r bon yl a n d 3.4 Å fr om H is57.177,178 H owever , t h is
posit ion is n or m a lly occu pied t h e P 2-P 3 r esidu es of
a pept ide su bst r a t e, wh ich su ggest s t h a t t h is wa t er
is n ot in volved in t h e h ydr olysis of pept idyl a cylen zym e. A bet t er ca n dida t e for t h e h ydr olyt ic wa t er
h a s been iden t ified in pept idyl a cylen zym es of SGP A
a n d ela st a se.39,42 Th is wa t er m olecu le is fou n d ∼3 Å
fr om H is57 a n d ∼3 Å fr om t h e ca r bon yl of t h e
a cylen zym e, a t a n opt im a l a n gle for n u cleoph ilic
a t t a ck fr om t h e lea vin g gr ou p side of t h e a ct ive sit e.
H owever , a s pr om isin g a s t h is ca n dida t e is, it m u st
be r em em ber ed t h a t t h ese com plexes a r e u n u su a lly
st a ble a cylen zym es a t a pH wh er e ca t a lysis does n ot
occu r r ea dily; t h e a ct u a l h ydr olyt ic wa t er m a y n ot
pr esen t .
5. Stereochemistry of the Tetrahedral Intermediate
The stereoelectronic requirements of forma tion a n d
br ea kdown of t h e t et r a h edr a l in t er m edia t e pose a n
Chemical Reviews, 2002, Vol. 102, No. 12 4511
a ddit ion a l con u n dr u m .62,173 Wh en a n u cleoph ile a t t a cks a n a m ide bon d, t h e lon e elect r on pa ir s of t h e
oxya n ion a n d t h e n it r ogen of t h e lea vin g gr ou p m u st
be a n t iper ipla n a r t o t h e n ew bon d (F igu r e 11). In a
ser in e pr ot ea se r ea ct ion , t h is st er eoch em ist r y lea ves
t h e lon e elect r on pa ir of t h e a m in e lea vin g gr ou p
poin t in g a wa y fr om H is57-H +. Th e n it r ogen m u st
u n der go a n in ver sion t o posit ion t h e lon e pa ir for
pr ot on a t ion . Alt h ou gh it is u n clea r h ow t h e pr ot ea se
will in flu en ce t h e in ver sion pr ocess, su ch in ver sion s
occur rea dily in solution. Perha ps the enzym e ca n use
in t er a ct ion s wit h t h e oxya n ion h ole a n d Sʹ′ sit es t o
over com e t h e st er eoelect r on ic im per a t ive of solu t ion
ch em ist r y.62
Figu re 11. Stereoelectronic mechanism for serine protease
ca t a lysis.
6. Stabilization of the Tetrahedral Intermediate/Strain in
the Acylenzyme Intermediate
En zym e ca ta lysis is m ost sim ply described in t erms
of t r a n sit ion st a t e st a biliza t ion , bu t in pr a ct ice
in t er a ct ion s t h a t st a bilize t h e t r a n sit ion st a t e will
a lso st r a in t h e gr ou n d st a t e. Th is idea ca n be
dem on st r a t ed by con sider in g t h e oxya n ion h ole,
wh er e in t er a ct ion s sh ou ld bot h st a bilize t h e t et r a h edr a l in t er m edia t e a n d a ct iva t e t h e su bst r a t e for
n u cleoph ilic a t t a ck.
St a biliza t ion of t et r a h edr a l in t er m edia t es is eviden t in t h e ch a r a ct er iza t ion of t r a n sit ion st a t e a n a logu e com plexes (F igu r e 6). Th e pK a of t h e h em iket a l
oxygen in ch lor om et h yl ket on e-in a ct iva t ed ch ym ot r ypsin is ∼2 u n it s lower t h a n t h e pK a in solu t ion ,170
wh ile t h e pK a of t h e h em iket a l oxygen of t r iflu or oket on e a ddu ct is >5 u n it s lower .149 Th ese r esu lt s
su ggest t h a t t h e oxya n ion h ole in t er a ct ion s st a bilize
t h e t et r a h edr a l in t er m edia t e by ∼6 kca l/m ol.
Un for t u n a t ely, dist or t ion of t h e pept ide su bst r a t e
h a s been difficu lt t o dem on st r a t e. H owever , con vin cin g eviden ce for su ch dist or t ion /a ct iva t ion of t h e
a cylen zym e com es fr om r eson a n ce Ra m a n spect r oscopy. Tonge and Carey measured the carbonyl stretching frequencies of a series of arylacryloyl acylenzymes
of ch ym ot r ypsin a n d su bt ilisin .140,179-181 Recen t wor k
ext en ds t h ese obser va t ion s t o p-(dim et h yla m in o)ben zoylen zym es.182 Mu lt iple fea t u r es a r e obser ved in
t h e ca r bon yl pr ofile of t h ese a cylen zym es, su ggest in g
t h a t t h e ca r bon yl gr ou p h a s m u lt iple con for m a t ion s.
Beca u se h ydr ogen bon din g decr ea ses t h e st r et ch in g
fr equ en cy of ca r bon yl bon d, t h e lowest fr equ en cy
ba n d wa s a ssign ed t o t h e con for m a t ion of t h e a cylenzyme with the carbonyl bound in the oxyanion hole.
Th is a ssign m en t is su bst a n t ia t ed by t h e followin g
obser va t ion s: (1) Th e in t en sit y of t h is ba n d in cr ea ses
wit h pH , displa yin g t h e sa m e pH depen den ce a s t h e
dea cyla t ion r a t e con st a n t .140 (2) Th is ba n d is a bsen t
in su bt ilisin va r ia n t s wh er e t h e oxya n ion h ole h a s
been elim in a t ed.179 Im por t a n t ly, t h e fr equ en cy of t h is
4512 Chemical Reviews, 2002, Vol. 102, No. 12
ba n d (νCdO ) cor r ela t es wit h dea cyla t ion r a t e for a
series of a cylenzym es. Th is cor r ela t ion spa n s 4 orders
of m a gn it u de in dea cyla t ion r a t e con st a n t s (6.8 ×
10 -6 -0.3 s -1 ).180,181 F u r t h er , νCdO ca n be r ela t ed t o
ca r bon yl bon d len gt h , in dica t in g t h a t ca r bon yl bon d
len gt h in cr ea ses 0.025 Å a s t h e dea cyla t ion r a t e
con st a n t in cr ea ses 16300-fold. Th is in cr ea se is ∼11%
of t h e ch a n ge in cu r r ed u pon t r a n sfor m a t ion of a
ca r bon yl gr ou p t o a C-O bon d. If t h is cor r ela t ion is
ext r a pola t ed t o t h e dea cyla t ion r a t es obser ved for
good su bst r a t es (∼100 s -1 ), t h e ca r bon yl bon d is
dist or t ed ∼14% t owa r d a C-O bon d. La st , m odel
syst em s in dica t e t h a t t h e ch a n ge in ∆H a ssocia t ed
wit h t h e sh ift s in νCdO is 6.4 kca l/m ol, sim ila r t o t h e
ch a n ges in t r a n sit ion st a t e st a biliza t ion of 5.7 kca l/
m ol over t h e r a n ge of com pou n ds. Th er e is su fficien t
en er gy in t h e h ydr ogen bon din g in t er a ct ion s of t h e
oxya n ion h ole t o pr ovide t h is t r a n sit ion st a t e st a biliza t ion . Sim ila r ly, m u lt iple est er ba n ds a r e a lso
obser ved in F ou r ier t r a n sfor m in fr a r ed spect r oscopy
of cin n a m oyl-ch ym ot r ypsin s, a n d h a ve likewise been
a t t r ibu t ed t o m u lt iple con for m a t ion s of t h e ca r bon yl
gr ou p.183 Th is wor k pr ovides fu r t h er eviden ce for
dist or t ion of t h e ca r bon yl bon d in t h e a cylen zym e.
In t er est in gly, t h e est er ca r bon yl fr equ en cy cor r ela t es
wit h dielect r ic con st a n t in m odel com pou n ds. Th is
cor r ela t ion su ggest s t h a t t h e ca r bon yl of t h e a cylen zym e exper ien ces a dielect r ic con st a n t of 70 in t h e
oxyanion hole conformation and 40 in the nonproduct ive con for m a t ion .
It a ppea r s t h a t t h e fu n ct ion of t h e ca t a lyt ic t r ia d
a n d oxya n ion h ole ca n be expla in ed by t h e elect r ost a t ic com plem en t a r it y of t h e a ct ive sit e a n d t r a n sit ion st a t e.167 Th e pr eor ga n iza t ion of t h e en zym e
a ct ive sit e pr ovides a su bst a n t ia l a dva n t a ge r ela t ive
t o t h e u n ca t a lyzed r ea ct ion wh er e solven t m olecu les
m u st or ga n ize t o st a bilize t h e developin g ch a r ges a s
t h e r ea ct ion pr oceeds.262 Wa r sh el h a s descr ibed t h is
ph en om en on a s solva t ion su bst it u t ion r a t h er t h a n
desolva t ion .167 Th is pr eor ga n iza t ion is pa id for by t h e
foldin g en er gy of t h e en zym e.263
IV. Kinetics of the Serine Protease Reaction
Th e ser in e pr ot ea se r ea ct ion is gen er a lly con sider ed t o h a ve t h r ee-st ep kin et ic m ech a n ism (sh own
in F igu r e 12): (a ) for m a t ion of a n en zym e-su bst r a t e
(E‚S) com plex (K s ) k -1/ k 1); (b) a cyla t ion of t h e a ct ive
sit e ser in e (k 2 ); a n d (c) h ydr olysis of t h e a cylen zym e
F ig u re 12. Th e kin et ic m ech a n ism of ser in e pr ot ea ses.
Hedstrom
in t er m edia t e (k 3 ). Alt h ou gh t h e t h r ee-st ep m ech a n ism of F igu r e 12 is widely a ccept ed, it is im por t a n t
t o n ot e t h a t som e of it s key fea t u r es h a ve n ot been
r igor ou sly t est ed. F ir st , bot h k 2 a n d k 3 a r e com posit e
rate constants that include a chemical transformation
a n d a pr odu ct dissocia t ion st ep. It is gen er a lly
a ssu m ed t h a t t h e pr odu ct dissocia t ion st ep is fa st
r ela t ive t o t h e ch em ica l t r a n sfor m a t ion . Th e eviden ce
u su a lly cit ed t o su ppor t t h is a ssu m pt ion is t h e
pr esen ce of a la r ge solven t kin et ic isot ope effect (∼3)
on k ca t , wh ich is con sist en t wit h r a t e-lim it in g ch em ical transformation.184 However, solvent isotope effects
ca n be difficu lt t o in t er pr et beca u se t h e solu bilit y of
su bst r a t es a n d pr odu ct s, t h e viscosit y of t h e solu t ion
a n d even t h e st r u ct u r e of t h e pr ot ein ca n ch a n ge in
D 2 O, so t h a t t h e isot ope effect m a y n ot be defin it ive
pr oof of r a t e-lim it in g ch em ist r y.185 Secon d, t h e m ech a n ism in dica t es t h a t dissocia t ion of t h e lea vin g gr ou p
m u st occu r pr ior t o h ydr olysis of t h e a cylen zym e.
Aga in , t h er e is lit t le eviden ce on t h is poin t . Acylt r a n sfer exper im en t s in dica t e t h a t h ydr olysis ca n
occu r fr om a n a cylen zym e-n u cleoph ile com plex,
wh ich a ppea r s t o con t r a dict t h is a ssu m pt ion .97,186-188
H owever , t h ese a cylen zym e-n u cleoph ile com plexes
m a y be n on pr odu ct ive com plexes t h a t do n ot for m
du r in g t h e n or m a l ca t a lyt ic cycle. F or exa m ple,
du r in g t h e h ydr olysis of a pept ide su bst r a t e, t h e
lea vin g gr ou p wou ld be con st r a in ed t o in t er a ct a t t h e
S1ʹ′ sit e. In con t r a st , a n a dded n u cleoph ile cou ld for m
a nonproductive complex at another Sʹ′ site that would
n ot im pede h ydr olysis of t h e a cylen zym e. Th u s, t h e
or der of h ydr olysis a n d pr odu ct r elea se is n ot well
est a blish ed.
Sever a l ot h er a ssu m pt ion s a r e fr equ en t ly m a de
wh en eva lu a t in g t h e kin et ics of ser in e pr ot ea se
r ea ct ion s. Th e Mich a elis-Men t en pa r a m et er s k ca t ,
K m , a n d k ca t /K m a r e com posit es of t h e r a t e con st a n t s
a s sh own in F igu r e 12. In pa r t icu la r , t h e expr ession
for K m includes k 3 as well as k 1, k -1, and k 2. Increasing
va lu es of k 2 will in cr ea se K m u n t il k 2 g k 3, bu t fu r t h er
in cr ea sin g k 2 will decr ea se K m (F igu r e 12), so t h a t
K m ca n be exceedin gly difficu lt t o in t er pr et wit h
r espect t o su bst r a t e a ffin it y. In a ddit ion , t h e expr ession s in F igu r e 12 a r e fr equ en t ly sim plified by
a ssu m in g k -1 . k 2. Th is a ssu m pt ion is pr oba bly va lid
for poor su bst r a t es, bu t t h e h ydr olysis of good su bst r a t es is oft en diffu sion con t r olled, wh ich in dica t es
k 2 g k -1 .189-191
Th e a sser t ion t h a t a cyla t ion is r a t e-lim it in g for
a m ide h ydr olysis, i.e., k 3 . k 2 , is by fa r t h e m ost
in discr im in a t ely a pplied a ssu m pt ion . Sem in a l wor k
fr om Ben der a n d co-wor ker s dem on st r a t ed t h a t k 2
is the ra te-limiting step for the hydrolysis of N -acetylL -t r ypt oph a n a m ide, wh ile k 3 is r a t e lim it in g for t h e
h ydr olysis of N -a cet yl-L -t r ypt oph a n est er s.59 Th ese
obser va t ion s h a ve been gen er a lized t o st a t em en t s
su ch a s “a cyla t ion is r a t e det er m in in g for a m ide
su bst r a t es a n d dea cyla t ion is r a t e det er m in in g for
est er su bst r a t es of ser in e pr ot ea ses”, a s m a y be fou n d
in m a n y t ext books. Th is st a t em en t h a s ga in ed wide
a ccept a n ce beca u se a m ides a r e in t r in sica lly m u ch
less r ea ct ive t h a n est er s. H owever , N -a cet yl-L -t r ypt oph a n a m ide is a ver y poor su bst r a t e for ch ym ot r ypsin ,
wit h k ca t /K m ) 3.6 M -1 s -1 a n d k ca t ) 0.026 s -1 .
Serine Protease Mechanism and Specificity
E n zym es oft en pr ocess poor su bst r a t es wit h differ en t
r a t e-det er m in in g st eps t h a n good su bst r a t es. Ch ym ot r ypsin a n d t r ypsin h ydr olyze pept ides wit h va lu es
of k ca t /K m in t h e r a n ge of ∼10 7 M -1 s -1 a n d k ca t ≈ 100
s -1.192,193 Mich a elis-Men t en pa r a m et er s of t h is or der
of m a gn it u de a r e t ypica l for t h e r ea ct ion s of ser in e
proteases; thrombin and tissue plasminogen activator
h ydr olyze t h eir n a t u r a l su bst r a t es wit h k ca t /K m ’s ≈
10 7 M -1 s -1 .194,195 E ven F a ct or D, a pr ot ea se wit h
n ot or iou sly low a ct ivit y t owa r d oligopept ide a m ide
a n d est er su bst r a t es, clea ves it s n a t u r a l pr ot ein
su bst r a t e wit h k ca t /K m g 2 × 10 6 M -1 s -1 .196,197 Given
t h a t t h e va lu es of k ca t /K m for pept ide h ydr olysis ca n
be 10 6 -fold gr ea t er t h a n t h e a m ide su bst r a t es u sed
in Ben der ’s wor k, t h e a cyla t ion st ep sh ou ld n ot be
a ssu m ed t o be gen er a lly r a t e-lim it in g.
In deed, sever a l exper im en t s su ggest t h a t k 2 is n ot
a lwa ys r a t e-lim it in g for a m ide h ydr olysis. Com pa r a ble va lu es of k ca t h a ve been r epor t ed for t h e
h ydr olysis of a n a logou s oligopept ide a m ides a n d
est er s.192,198-201 Su ch obser va t ion s gen er a lly su ggest
t h a t a com m on st ep is r a t e-lim it in g, i.e., h ydr olysis
of t h e a cylen zym e in t er m edia t e. H owever , in fer r in g
in dividu a l r a t e con st a n t s fr om st ea dy-st a t e pa r a m et er s is a pr eca r iou s pr a ct ice. Th e m ost con vin cin g
ca se for r a t e-lim it in g dea cyla t ion in a m ide h ydr olysis
is t h e obser va t ion of bu r st s of pr odu ct for m a t ion
u n der pr e-st ea dy st a t e con dit ion s. Su ch bu r st s a r e
obser ved in t h e h ydr olysis p-n it r oa n ilida m ides by
h u m a n leu kocyt e ela st a se a n d in t h e h ydr olysis of
m et h ylcou m a r in a m ides by Kex2 pr ot ea se a n d
fu r in .202-204 Sim ila r bu r st s h a ve been obser ved in t h e
h ydr olysis of pept ide m et h ylcou m a r in a m ides by
trypsin (X. Liu and L. Hedstrom, unpublished experim en t s). Most con vin cin gly, a bu r st is obser ved in t h e
h ydr olysis of a pept ide su bst r a t e by Kex2.205 Th u s,
t h e gen er a liza t ion “k 2 is r a t e det er m in in g for t h e
h ydr olysis of a m ide su bst r a t es by ser in e pr ot ea ses”
is n ot wa r r a n t ed.
Th e a bove wor k is fr equ en t ly cr it icized beca u se t h e
p-n it r oa n ilides a n d m et h ylcou m a r in a m ides lea vin g
gr ou ps a r e m or e r ea ct ive t h a n a pept ide bon d.
H owever , a m ide h ydr olysis h a s lit t le depen den ce on
lea vin g gr ou p (βlg ) 0.07) a n d t h e plot of t h e va lu es
of k OH for t h e h ydr olysis of bot h a m ides a n d a n ilides
fa ll on a sin gle lin e wh en plot t ed a ga in st pK a of t h e
lea vin g gr ou p.206 Wh ile p-n it r oa n ilides a r e except ion a lly r ea ct ive in solu t ion ,207 t h is u n u su a l r ea ct ivit y is
n ot r eca pit u la t ed in pr ot ea se r ea ct ion s. Th e h ydr olysis of a n ilides, in clu din g p-n it r oa n ilides, by ch ym ot r ypsin displa ys n o depen den ce on t h e pK a of t h e
lea vin g gr ou p (m ost likely du e t o gen er a l ba se
ca t a lysis). Mor eover , pept ide bon ds a r e m ore r ea ct ive
t h a n a n ilides wit h lea vin g gr ou ps of com pa r a ble
pK a .110 Th er efor e, it is u n likely t h a t t h e r esu lt s cit ed
a bove ca n be a t t r ibu t ed t o t h e u n u su a l r ea ct ivit y of
t h e lea vin g gr ou ps. Th ese obser va t ion s fu r t h er dem on st r a t e t h a t t h e r a t e-det er m in in g st ep of a n a m ide
su bst r a t e sh ou ld n ot be a u t om a t ica lly a ssign ed t o
a cyla t ion .
Sever a l m et h ods h a ve been u sed t o m ea su r e t h e
va lu es of t h e in dividu a l r a t e con st a n t s k 1, k -1, k 2, a n d
k 3. Su r pr isin gly, a lt h ou gh pr e-st ea dy st a t e a n d sin gle
t u r n over exper im en t s a r e t h e best m et h ods for de-
Chemical Reviews, 2002, Vol. 102, No. 12 4513
t er m in in g in dividu a l r a t e con st a n t s, t h ey h a ve on ly
r a r ely been a pplied t o ser in e pr ot ea ses, a n d u su a lly
con fin ed t o t h e a n a lysis of est er h ydr olysis (for
exa m ples, see r efs 208-211). A r ecen t st u dy h a s
a pplied t h ese m et h ods t o t h e a n a lysis of t h e h ydr olysis of pept ide p-n it r oa n ilides by ela st a se.202 Th e
in dividu a l r a t e con st a n t s a r e m or e com m on ly det er m in ed by ch a r a ct er izin g t h e h ydr olysis of a n a logou s
a m ide/est er pa ir s.59,199,200 Dea cyla t ion is u su a lly r a t elim it in g in t h e h ydr olysis of est er s, so t h a t k ca t ,est er )
k 3 . Wh ile t h is fa ct is oft en t a ken for gr a n t ed, it ca n
be su bst a n t ia t ed if t h e r ea ct ion s of a set of a n a logou s
est er s wit h differ en t lea vin g gr ou ps h a ve t h e sa m e
k ca t ; t h is obser va t ion su ggest s t h a t a com m on st ep is
r a t e-lim it in g, i.e., dea cyla t ion . Likewise, a n a n a logou s a m ide su bst r a t e m u st for m t h e sa m e a cylen zym e, so t h a t k 3 m u st be t h e sa m e a s in t h e est er
r ea ct ion . Th e va lu es of K s a n d k 2 ca n t h en be
det er m in ed fr om t h e equ a t ion s in F igu r e 12 (wit h t h e
a ssu m pt ion t h a t k -1 . k 2 ). Th e va lu es of K s , k 2 , a n d
k 3 ca n a lso be det er m in ed u sin g a cylt r a n sfer r ea ct ion s u n der con dit ion s wh er e t h e a dded n u cleoph ile
does n ot for m a com plex wit h t h e a cylen zym e.94,212
Th is m et h od is especia lly u sefu l in eva lu a t in g t h e
h ydr olysis of su bst r a t es wh er e k 2 g k 3 . Th e a n a lysis
of t h e viscosit y depen den ce of k ca t /K m ca n yield va lu es
of k 1 a n d t h e r a t io of k -1 /k 2 .189 Th is m et h od m u st be
a pplied wit h ca u t ion beca u se it in volves ch a n gin g
solven t , wit h a t t en den t ch a n ges in dielect r ic con st a n t
a n d t h e possibilit y of ot h er n on specific effect s, a n d
t h er efor e r equ ir es ca r efu l a t t en t ion t o con t r ols. In dividu a l r a t e con st a n t s h a ve a lso been det er m in ed
by a n a lyzin g t h e t em per a t u r e depen den ce of t h e
Mich a elis-Men t en pa r a m et er s.191 Th is m et h od ca n n ot iden t ify t h e r a t e-lim it in g st ep a n d gen er a lly
oper a t es u n der t h e a ssu m pt ion t h a t k 3 . k 2 . Usin g
t h ese va r iou s m et h ods, va lu es of k 2 of 10 2 -10 4 s -1
for good est er su bst r a t es a n d 10-1000 s -1 for good
a m ide su bst r a t es h a ve been r epor t ed. Th e va lu e of
k 3 is u su a lly ∼50 s -1 for good su bst r a t es. Obviou sly,
t h e va lu es of k 2 a n d k 3 ca n be m u ch lower if t h e
su bst r a t e is n ot opt im a l. Th e va lu es of k 1 a n d k -1 a r e
typically 10 6-10 8 M -1 s -1 and 5-500 s -1, respectively.
V. Substrate Discrimination during Catalysis
A. The Hallmarks of Serine Protease Specificity
Specificit y is defin ed by h ow su bst r a t es com pet e
for a n en zym e, a n d is m ea su r ed by t h e va lu e of k ca t /
K m . Th e P 1/S1 in t er a ct ion con t r ols specificit y, a s
illu st r a t ed wit h t r ypsin , wh er e t h e va lu e of k ca t /K m
va r ies over 10 5 -fold a s t h e P 1/S1 in t er a ct ion is
opt im ized (Ta ble 3). Th is in cr ea se r esu lt s m a in ly
fr om a n in cr ea se in k ca t (10 4 -fold) r a t h er t h a n K m (10fold). In t er a ct ion s a t t h e S2-Sn sit es a lso st r on gly
in flu en ce specificit y, a s illu st r a t ed wit h ela st a se
wh er e t h e va lu e of k ca t /K m in cr ea ses 10 5 -fold a s t h e
a ddit ion a l P r esidu es a r e a dded (Ta ble 3). Aga in , t h e
in cr ea se r esu lt s m a in ly fr om a n in cr ea se in k ca t (10 3 fold) r a t h er t h a n K m (100-fold). Sim ila r ly, P ʹ′/Sʹ′ in t er a ct ion s in cr ea se k cat /K m , on ce a ga in via a n in cr ea se
in k ca t (Ta ble 3). Th e effect of t h e P ʹ′/Sʹ′ in t er a ct ion s
ca n be m ost dr a m a t ica lly illu st r a t ed by t h e in a ct iva t ion of ch ym ot r ypsin by ca r bon a t e est er s, wh er e
4514 Chemical Reviews, 2002, Vol. 102, No. 12
Hedstrom
Ta ble 3. Th e Hy d ro ly s is o f S u bs tra te s by Ra t Try p s in a n d P o rc in e P a n c re a tic Ela s ta s e
k ca t /K m (M -1 s -1 )
su bst r a t e
k ca t (s -1 )
K m (µM)
Tr ypsin b
Ra t
1.3 × 10 6
1.2 × 10 6
3
8
Su c-Ala -Ala -P r o-Ar g-AMC
Su c-Ala -Ala -P r o-Lys-AMC
Su c-Ala -Ala -P r o-Tyr -AMC
Su c-Ala -Ala -P r o-P h e-AMC
Ac-Ala -NH 2 c
Ac-P r o-Ala -NH 2 c
Ac-Ala -P r o-Ala -NH 2 d
Ac-P r o-Ala -P r o-Ala -NH 2 d
Ac-Ala -P r o-Ala -Ala -NH 2 d
Ac-P r o-Ala -P r o-Ala -Ala -NH 2 d
P or cin e P a n cr ea t ic E la st a se
<0.05
0.07
25
3700
520
25,500
Cbz-Va l-pNP
Cbz-P h e-pNP
Boc-Ala -Ala -Va l-SBzl
Boc-Ala -Ala -P h e-SBzl
H u m a n Leu kocyt e E la st a se e
480
65
3.0 × 10 6
<10
91
120
6.3 × 10 -4
6.3 × 10 -3
70
100
200
780
<0.008
0.007
0.1
9.3
2.4
37
160 (K i)
100
4.2
2.5
4.6
1.4
a
“-” den ot es clea ved bon d; AMC, a m in om et h ylcou m a r ide; pNP , p-n it r oph en yl; SBzl, t h ioben zyl.
fr om r ef 260. d Da t a fr om r ef 261. e Da t a fr om r ef 215.
Ta ble 4. In h ibitio n o f Ch y m o try p s in by Ca rbo n a te
Es te rs a
a
k on (M -1 s -1 )
OCH 3
O-Ala -OCH 2 CH 3
O-Ala -Leu -OCH 3
O-Ala -Leu -Ar g-OCH 3
9
200
2,400
>23,000
Da t a fr om r ef 213.
in t er a ct ion s wit h S1ʹ′-S3ʹ′ sit es in cr ea se t h e r a t e of
in a ct iva t ion >2000-fold (Ta ble 4).213
Im por t a n t ly, t h e S1 sit e a n d t h e r em ot e bin din g
sit es a ct in con cer t t o det er m in e ser in e pr ot ea se
specificit y.200,214,215 Wh ile t h e va lu e of k ca t /K m in cr ea ses dr a m a t ica lly wit h pept ide len gt h , t h is in cr ea se r equ ir es t h e cor r ect P 1-S1 in t er a ct ion .52,216
Th is ph en om en on ca n be illu st r a t ed wit h t h e h ydr olysis of p-n it r oph en ylest er s by h u m a n leu kocyt e
ela st a se (H LE ) (Ta ble 3).215 H LE displa ys lit t le
specificit y in t h e h ydr olysis of sin gle r esidu e est er s,
wit h t h e P 1-Va l su bst r a t e pr efer r ed by on ly 7-fold
over t h e P 1-P h e, even t h ou gh t h e S1 sit e of H LE
sh ou ld n ot a ccom m oda t e la r ge r esidu es. Th e a ddit ion
of t wo pept ide r esidu es in cr ea ses t h e h ydr olysis of
t h e P 1-Va l su bst r a t e by >10 3 , bu t d ecreases t h e
h ydr olysis of t h e P h e-con t a in in g su bst r a t e. Likewise,
in cr ea sin g pept ide len gt h dr a m a t ica lly in cr ea ses t h e
va lu e of k ca t /K m in t h e t r ypsin -ca t a lyzed h ydr olysis
of su bst r a t es con t a in in g P 1-Ar g or Lys, bu t n ot P 1P h e.200 Sim ila r r esu lt s a r e obser ved for ot h er ser in e
pr ot ea ses.
B. Specificity Is Determined by the Binding and
Acylation Steps
As noted a bove, k cat /K m depen ds on ly on t h e bin din g
a n d a cyla t ion st eps k 1 , k -1 , a n d k 2 (F igu r e 12). F r om
a n in t u it ive per spect ive, if t wo su bst r a t es com pet e
for pr ot ea se, t h e com pet it ion is “won ” wh en a cyla t ion
Da t a fr om r ef 218. c Da t a
Ta ble 5. Th e S p e c ific ity o f B in d in g , Ac y la tio n , a n d
D e a c y la tio n a
su bst r a t e
lea vin g gr ou p X
b
Ks
k ca t /K m
(M -1 s -1 ) (µM)
k2
(s -1 )
k3
(s -1 )
H u m a n Leu kocyt e E la st a se b
MeOSu c-Va l-pNA
75
788
MeOSu c-P r o-Va l-pNA
575
722
MeOSu c-Ala -P r o-Va l-pNA
56,000
810
0.06
0.42
43
10
11
13
Ra t Tr ypsin II
Z-Ar g-AMC c
4000
Tos-Gly-P r o-Ar g-AMC c
1 × 10 7
Z-Lys-AMC c
1500
D -Va l-Leu -Lys-AMC c
30,000
Ac-P h e-NH 2 d
0.2
Su c-Ala -Ala -P r o-P h e-AMC d 6
0.7
g1300
0.32
280
0.0002
0.007
65
56
130
18
30
60
96
g50
220
6500
1000
1000
a
Mech a n ist ic r a t e con st a n t s a s defin ed in F igu r e 12. “-”
denotes cleaved bond; pNA, p-nitroanilide; AMC, aminomethylcouma ride. b Da ta from ref 52. c Da ta from ref 243. d Data from
r ef 200.
occu r s a n d dea cyla t ion is ir r eleva n t . Sin ce t h e in cr ea se in t h e va lu es of k ca t /K m is der ived fr om a n
increa se in k cat rather than a decrease in K m , specificit y m u st be pr im a r ily det er m in ed by a cyla t ion . Th is
con clu sion is bor n e ou t wh en t h e st ea dy-st a t e kin et ic
pa r a m et er s a r e decon volu t ed in t o K s a n d k 2 (Ta ble
5). Th e va lu e of k 2 va r ies by m or e t h a n 10 3 × bet ween
good a n d poor su bst r a t es wh ile on ly 10-100-fold of
su bst r a t e discr im in a t ion der ives fr om su bst r a t e a ffin it y. 52,200,202,216 It is especia lly n ot ewor t h y t h a t lon g
pept ide su bst r a t es bin d wit h a ppr oxim a t ely t h e sa m e
a ffin it y a s sh or t su bst r a t es.
Alt h ou gh k 3 is n ot a com pon en t of k ca t /K m , it sh ou ld
n ot be com plet ely ign or ed. If t h e va lu e of k 3 is ver y
sm a ll, t h e a cylen zym e will be ver y st a ble, su bst r a t e
t u r n over will be ver y slow a n d t h e en zym e will be
essentially inactivated. The deacylation step is therefor e a cr it ica l det er m in a n t of su bst r a t e t u r n over . Th e
dea cyla t ion st ep a lso va r ies a m on g su bst r a t es, a lthough much more dramatic changes in the substrate
a r e r equ ir ed t o a ffect k 3 . In t er est in gly, wh ile t h e
va lu es of k 2 for P 1-Ar g con t a in in g su bst r a t es a r e
100-10 5-fold gr ea t t h a n P 1-P h e, t h e va lu es of K s a r e
<10-fold less a n d t h e va lu es of k 3 a r e sim ila r (Ta ble
5). Thus, the preferences of the binding, acylation and
Serine Protease Mechanism and Specificity
dea cyla t ion st eps a r e differ en t even t h ou gh t h ese
st eps occu r on t h e sa m e en zym e su r fa ce.
C. The Contribution of Substrate Association to
Specificity
In spect ion of t h e a lgebr a ic expr ession for k ca t /K m
r evea ls a n ot h er im por t a n t m ech a n ist ic fa ct : a s t h e
va lu e of k 2 in cr ea ses, k -1 will becom e in sign ifica n t ,
k ca t /K m ) k 1 a n d t h e r ea ct ion will be diffu sion con t r olled. If t h e va lu e of k 1 wa s det er m in ed by t h e
sim ple collision of su bst r a t e a n d pr ot ea se, t h en k 1
wou ld be sim ila r for a ll su bst r a t es, i.e., ∼10 8 M -1 s -1 .
H owever , va lu es of k 1 of 10 6 -10 7 M -1 s -1 h a ve been
r epor t ed, wh ich su ggest t h a t su bst r a t e a ssocia t ion is
m or e com plica t ed t h a n a sim ple collision . Obviou sly,
t h e su bst r a t e/pr ot ea se in t er fa ce h a s m a n y h ydr ogen
bon din g a n d va n der Wa a ls in t er a ct ion s wh ich seem
u n likely t o for m in a sin gle st ep. Som e eviden ce for
isom er iza t ion st eps h a s been r epor t ed.52,56 On e m igh t
expect t h a t t h e va lu e of k 1 sh ou ld decr ea se wit h
pept ide len gt h sin ce m or e in t er a ct ion s m u st for m .
Th e va lu e of k -1 sh ou ld a lso decr ea se, sin ce m or e
in t er a ct ion s wou ld h a ve t o be disr u pt ed wh en t h e
su bst r a t e dissocia t es. If t h e va lu es of k 1 a n d k -1
decr ea se in pa r a llel wit h pept ide len gt h , t h en t h e
over a ll a ffin it y will r em a in con st a n t . Th is idea ca n
be illu st r a t ed wit h t h e in h ibit ion of ch ym ot r ypsin by
t r iflu or oket on es. Ac-Leu -P h e-TF K is a m or e pot en t
in h ibit or of ch ym ot r ypsin t h a n Ac-P h e-TF K (K i ) 0.5
a n d 4.5 n M, r espect ively; va lu es a r e cor r ect ed for fr ee
ketone concentra t ion ).49 Ac-Leu -P h e-TF K h a s a lower
va lu e of k 1 t h a n Ac-P h e-TF K (4 × 10 6 a n d 3 × 10 7
M -1 s -1 , r espect ively), a n d a lower va lu e of k -1 (0.002
a n d 0.13 s -1 , r espect ively). Th e st r u ct u r e of t h e
ch ym ot r ypsin does n ot ch a n ge a ppr ecia bly in t h e t wo
in h ibit or com plexes, su ggest in g t h e kin et ics of AcLeu -P h e-TF K in h ibit ion a r e slower beca u se m or e
en zym e-in h ibit or con t a ct s m u st for m .74
Wh en a r ea ct ion is diffu sion con t r olled, t h e con forma tion a nd solva t ion of t h e su bst r a t e a n d protea se
will det er m in e t h e r a t e of su bst r a t e a ssocia t ion , a n d
h en ce of specificit y. Th ese con sider a t ion s illu st r a t e
wh y t h e st r u ct u r es of bot h fr ee en zym e an d free
su bstrate m u st be kn own t o a ssess t h e ba sis of
specificit y. Un for t u n a t ely, t h e st r u ct u r e of t h e su bst r a t e in solu t ion is r a r ely con sider ed in a n y en zym a t ic r ea ct ion . Sever a l pu zzlin g obser va t ion s a bou t
ser in e pr ot ea se specificit y m igh t be expla in ed by t h e
solu t ion con for m a t ion of pept ide su bst r a t es. F or
exa m ple, t h e P 3 r esidu e a ppea r s t o be a m a jor
det er m in a n t of specificit y even wh en it m a kes few
con t a ct s wit h t h e en zym e. La r ge P 3 r esidu es m a y
fa vor a m or e ext en ded pept ide con for m a t ion t h a t
wou ld fa vor a ssocia t ion . Th e su r pr isin g obser va t ion
t h a t t h e su bst it u t ion of est er bon ds a t t h e P 4-P 3 a n d
P 3-P 2 a m ide bon ds h a s lit t le effect on t h e va lu e of
k ca t /K m in t r ypsin r ea ct ion s 46 m igh t a lso be expla in ed
by ch a n ges in solu t ion con for m a t ion a n d/or solva t ion .
D. Implications for the Specificity of Ester
Hydrolysis
Th e specificit y of est er h ydr olysis m a y be dom in a t ed by su bst r a t e a ssocia t ion . On ce bou n d in t h e
Chemical Reviews, 2002, Vol. 102, No. 12 4515
a ct ive sit e cleft , t h e in t r in sic r ea ct ivit y of a n est er
en su r es a cyla t ion a n d pr ogr ession t h r ou gh t h e ca t a lyt ic cycle. Th is con dit ion a lm ost cer t a in ly h olds for
p-n it r oph en ylest er s, wh er e a cyla t ion is so fa cile t h a t
t h e r ea ct ion is con cer t ed. Th u s, t h e specificit y of est er
h ydr olysis will u n der est im a t e t h e pot en t ia l of a
pr ot ea se t o discr im in a t e bet ween pept ide su bst r a t es.
Th e va lidit y of t h is idea is illu st r a t ed in t h e h ydr olysis of “in ver se su bst r a t es”. Tr ypsin h ydr olyzes pa m idin oph en yl a cet a t e, wh er e t h e lea vin g gr ou p
bin ds in t h e S1 sit e, a lm ost a s well a s n or m a l est er
su bst r a t es (k ca t /K m ) 10 5 ver su s 10 7 M -1 s -1 ) (F igu r e
13).217 Sim ila r ly, t h e fa cile r ea ct ion of p-n it r oph en yl
gu a n idin oben zoa t e wit h ch ym ot r ypsin ca n a lso be
expla in ed by a n in ver se or ien t a t ion , wh er e t h e pn it r oph en yl lea vin g gr ou p bin ds in t h e S1 sit e a n d
t h e gu a n idin o gr ou p in t er a ct s wit h S1ʹ′.
F ig u re 13. In ver se su bst r a t es of t r ypsin .
Wh en t h e r ea ct ion is diffu sion con t r olled, t h e r a t elim it in g t r a n sit ion st a t e will n ot in volve for m a t ion
of t h e t et r a h edr a l in t er m edia t e a s gen er a lly su pposed; instea d the ra te-limiting step will be formation
of t h e E ‚S com plex. Th e gen er a l fea t u r es of t h is
transition state might include the canonical substrate
con for m a t ion a s well a s in t er a ct ion s a t t h e va r iou s
su bsit es. Ma n y in vest iga t or s h a ve a ssu m ed t h a t
pr ot ein in h ibit or s, wh ich a r e locked in t h e ca n on ica l
con for m a t ion , for m com plexes t h a t m im ic gr ou n dst a t e E ‚S com plexes. H owever , t h e va lu es of K i for
pr ot ein in h ibit or s cor r ela t e wit h va lu es of k ca t /K m for
th e a n a logou s su bstra tes, a fea tu re of tra n sit ion st a t e
a n a logu es bu t n ot gr ou n d-st a t e in h ibit or s.38 Th e
ca n on ica l con for m a t ion m a y be a h igh en er gy con for m a t ion t h a t a ct iva t es t h e scissile bon d a n d t h u s
m a y be t h e t r a n sit ion st a t e-like fea t u r e of pr ot ein
in h ibit or s.
VI. Redesigning the
Trypsin−Chymotrypsin−Elastase Paradigm
Text books oft en illu st r a t e en zym e specificit y wit h
t h e pa n cr ea t ic ser in e pr ot ea ses t r ypsin , ch ym ot r ypsin , a n d ela st a se: t r ypsin , wit h Asp189, clea ves
after P1 Arg/Lys residues; chymotrypsin, with Ser189,
clea ves a ft er P 1 P h e/Tyr /Tr p; a n d ela st a se, wit h
Va l216 a n d Th r 226, clea ves a ft er P 1 Ala /Va l. Th e
t r ypsin -ch ym ot r ypsin -ela st a se pa r a digm su ggest s
t h a t pr ot ea se specificit y is det er m in ed by a few
st r u ct u r a l elem en t s. H owever , t h e obviou s m u t a t ion s
fa il t o t r a n sfer t h e specificit y of on e pr ot ea se in t o
a n ot h er .
A. Redesigning Amidase Activity
Swit ch in g t h ese elem en t s fr om on e fr a m ewor k t o
t h e n ext sh ou ld be su fficien t t o ch a n ge specificit y.
H owever , t h e su bst it u t ion of Asp189 wit h Ser does
n ot con ver t t r ypsin in t o pr ot ea se wit h specificit y for
P 1-P h e (Ta ble 6).200,218,219 Tr ypsin does a cqu ir e ch ym ot r ypsin -like specificit y wh en t h e r esidu es of t h e
S1 sit e a n d t wo su r fa ce loops a r e r epla ced wit h t h eir
4516 Chemical Reviews, 2002, Vol. 102, No. 12
Hedstrom
Ta ble 6. Me c h a n is tic Kin e tic P a ra m e te rs fo r Am id e
Hy d ro ly s is by Ch y m o try p s in , Try p s in , a n d Try p s in
Mu ta n ts a
su bst r a t e
k ca t /K m
(M -1 s -1 )
Ks
(M)
k2
(s -1 )
Ch ym ot r ypsin
Ac-P h e-NH 2
18
0.023
0.43
Su c-Ala Ala P r oP h e-AMC 4.9 × 10 5 2.5 × 10 -4 12
Su c-Ala Ala P r oP h e-pNA 5.6 × 10 5 1.5 × 10 3 850
k3
(s -1 )
60
52
52
Tr ypsin
Ac-P h e-NH 2
0.2
1 × 10 -3
2 × 10 -4
Su c-Ala Ala P r oP h e-AMC 6.0
1.1 × 10 -3 0.007
Su c-Ala Ala P r oP h e-pNA 9
g 0.25
g 0.2
30
36
36
D189S
Ac-P h e-NH 2
0.1
0.17
0.018
Su c-Ala Ala P r oP h e-AMC 38
2.8 × 10 -3 0.10
Su c-Ala Ala P r oP h e-pNA 21
0.15
0.29
39
33
33
Tr f Ch [S1+L1+L2]
Ac-P h e-NH 2
0.3
0.065
Su c-Ala Ala P r oP h e-AMC 800
>0.006
Su c-Ala Ala P r oP h e-pNA 1.7 × 10 3 0.011
Tr f Ch [S1+L1+L2+Y172W]
Ac-P h e-NH 2
0.7
g0.1
Su c-Ala Ala P r oP h e-AMC 9.3 × 10 3 g0.006
Su c-Ala Ala P r oP h e-pNA 8.3 × 10 4 5 × 10 -4
a
0.018
>4
20
33
37
37
g0.08
g60
41
38
63
63
Da t a fr om r ef 220.
Figu re 14. The transfer of P1-Phe specificity into trypsin.
Th e va lu es of K s , k 2 , a n d k 3 a r e plot t ed ver su s k ca t K m
for t h e h ydr olysis of Su c-Ala -Ala -P r o-P h e-AMC by
t r ypsin , Asp189Ser t r ypsin , Tr f Ch [S1+L1+L2], Tr f
Ch [S1+L1+L2+Y172W] a n d ch ym ot r ypsin . Th e a cyla t ion
r a t e k 2 in cr ea ses a s k ca t /K m in cr ea ses, bu t t h e va lu es of K s
a n d k 3 r em a in con st a n t .
cou n t er pa r t s fr om ch ym ot r ypsin . Th ese su bst it u t ion s
a r e Asp189Ser , Gln 192Met , in ser t ion of Th r 219,
Ile138Th r , t h e 185 loop (r esidu es 185-188) a n d t h e
220 loop (r esidu es 221-224). Th is m u t a n t pr ot ea se,
Tr f Ch [S1+L1+L2], h a s 100× m or e a ct ivit y t h a n
D189S, a n d h a s 0.5% of t h e a ct ivit y of ch ym ot r ypsin
(Ta ble 6). An a ddit ion a l su bst it u t ion , Tyr 172Tr p,
in cr ea ses a ct ivit y t o wit h in 15% of ch ym ot r ypsin .220
The insta lla tion of chymotrypsin-like a ctivity in to th e
t r ypsin fr a m ewor k is a lm ost en t ir ely t h e r esu lt of
a cceler a t ion of t h e a cyla t ion st ep, wh ich in cr ea ses by
∼10 4 -fold (F igu r e 14). Dea cyla t ion is com pa r a ble in
a ll of t h e en zym es. H owever , n on e of t h e m u t a n t
pr ot ea ses bin d su bst r a t es well.
Alt h ou gh t h ese m u t a t ion s focu sed on t h e S1 sit e,
t h e in cr ea se in a cyla t ion or igin a t es in t h e in t er a ct ion s of t h e m or e r em ot e sit es. F or ch ym ot r ypsin , t h e
va lu e of k 2 is ∼400-fold la r ger for a lon g su bst r a t e
t h a n a sh or t su bst r a t e. In con t r a st , for D189S
t r ypsin , t h e va lu es of k 2 for t h e h ydr olysis of
sh or t a n d lon g su bst r a t es a r e sim ila r (Ta ble 6).
Th u s, u n like ch ym ot r ypsin , D189S t r ypsin ca n n ot
u t ilize bin din g in t h e r em ot e sit es t o in cr ea se a cyla t ion . H owever , in Tr f Ch [S1+L1+L2] a n d Tr f
Ch[S1+L1+L2+Y172W], acylation increases by >10 3fold between short a nd long substra tes. Thus, t he 185
a n d 220 loops m edia t e t h e t r a n sla t ion of r em ot e
bin din g in t er a ct ion s in t o ca t a lysis.
Wh y is Asp189Ser t r ypsin a poor n on specific pr ot ea se? F ir st , t h e su bst it u t ion of Asp189 wit h Ser
defor m s t h e S1 sit e a n d t h e a ct iva t ion dom a in (t h e
r egion t h a t r ea r r a n ges du r in g zym ogen a ct iva tion).219,221 This is the all too typical protein engineerin g r esu lt : m u t a t ion lea ds t o u n a n t icipa t ed defect s
in st r u ct u r e. In t er est in gly, BP TI in du ces D189S t o
a ssu m e a t r ypsin -like con for m a t ion , m a skin g t h ese
st r u ct u r a l defect s 222 a n d u n der scor in g t h e n eed t o
evaluate both free and bound enzymes and substrates
when assessing the structural basis of specificity. The
su bst it u t ion of Asp189 wit h H is defor m s t h e S1 sit e
even in t h e pr esen ce of BP TI, fu r t h er illu st r a t in g t h e
in h er en t fr a gilit y of t h e S1 sit e.223 Th ese obser va t ion s
su ggest t h a t m u t a t ion of Asp189 m igh t be su fficien t
t o ch a n ge specificit y if on ly t h e S1 sit e wa s m or e
st r u ct u r a lly sou n d.
H ow do t h e 185 a n d 220 loops a n d Tyr 172Tr p
su bst it u t ion in flu en ce ca t a lysis? Th e Tyr 172Tr p m u t a t ion h elps r ebu ild t h e S1 sit e in t h e pr esen ce of
Ser 189. Th e S1 sit e of Tr f Ch [S1+L1+L2+Y172W],
u n like D189S t r ypsin or Tr f Ch [S1+L1+L2], is
sufficiently structured to a llow profla vin binding. The
st r u ct u r es of t h e ch lor om et h yl ket on e-in a ct iva t ed
com plexes of t h ese pr ot ea ses a lso su ggest t h a t t h e
Tyr 172Tr p m u t a t ion st a bilizes t h e S1 sit e.16 Over a ll,
t h e st r u ct u r es of bot h Tr f Ch [S1+L1+L2] a n d Tr
f Ch [S1+L1+L2+Y172W] r esem ble ch ym ot r ypsin ,
bu t t h e 185 a n d 220 loops a r e disor der ed in bot h
m u t a n t s. Tr f Ch [S1+L1+L2+Y172W] is m or e
st r u ct u r ed t h a n Tr f Ch [S1+L1+L2], in keepin g
wit h it s h igh er a ct ivit y. Th e su bst it u t ion of t h e 185
a n d 220 loops a r e pr oba bly r equ ir ed t o con st r u ct a n
S1 sit e con t a in in g Ser 189.
Th e fr a gilit y of t h e S1 sit e is n ot t h e wh ole st or y.
Th e su bst it u t ion of t h e 185 a n d 220 loops h a ve a
su bt le effect on t h e polypept ide bin din g sit e: t h e
con for m a t ion of Gly216 ch a n ges so t h a t t h e ca r bon yl
poin t s t owa r d t h e P 3 NH in a bet t er or ien t a t ion for
t h e h ydr ogen bon d. Th e fu n ct ion of t h e polypept ide
bin din g sit e m a y be m edia t ed by t h e con for m a t ion
of Gly216, a n d t h er efor e by t h e 185 a n d 220 loops.15
Th e loops m a y a lso m odu la t e t h e dyn a m ic pr oper t ies
of t h e a ct ive sit e cleft . Gly216 a n d t h e a n a logou s 220
loop a lso a ppea r t o be im por t a n t det er m in a n t s of
alpha lytic protease specificity, where strong evidence
exist s for t h e in volvem en t of dyn a m ic pr ocesses (see
below).224,225
Th e S1 sit e, 185 a n d 220 loops, a n d r esidu e 172 do
n ot defin e a u n iver sa l set of st r u ct u r a l det er m in a n t s
of specificit y in ser in e pr ot ea ses. Th is set of su bst it u t ion s fa ils t o in st a ll t r ypsin -like specificit y in t o ch ym ot r ypsin ,226 n or do t h ey in st a ll a n ela st a se-like
specificit y in t o t r ypsin .227 Th ese r esu lt s su ggest t h a t
a differ en t st r u ct u r a l solu t ion is r equ ir ed for ea ch
Serine Protease Mechanism and Specificity
specificit y. In a ddit ion , a lt er n a t e solu t ion s t o t h e
t r ypsin -t o-ch ym ot r ypsin con ver sion m a y exist . St a biliza t ion of t h e a ct iva t ion dom a in wou ld a lso st a bilize t h e S1 sit e. Ba ct er ia l ser in e pr ot ea ses do n ot u se
t h e zym ogen a ct iva t ion m ech a n ism of m a m m a lia n
serine proteases, which suggests tha t other stra tegies
exist for st a bilizin g t h e S1 sit e. Th is st r a t egy is
pr oba bly t h e m ech a n ism oper a t in g a m et a l-a ct iva t ed
Asp189Ser trypsin variant. The substitution of Asn143
a n d Glu 151 wit h H is cr ea t es a m et a l bin din g sit e in
t r ypsin .228 Th is m et a l sit e ca n a lso in t er a ct wit h a
P 2ʹ′ H is r esidu e, t h u s in st a llin g specificit y for H is a t
t h e S2ʹ′ sit e. Th ese m u t a t ion s a lso in cr ea se t h e
activity of Asp189Ser trypsin by 300-fold. Asn143 and
Glu 151 a r e a lso pa r t of t h e a ct iva t ion dom a in ; m et a l
bin din g will st a bilize t h e a ct iva t ion dom a in , a n d by
extension, the S1 site, which accounts for the increase
in a ct ivit y.
Un like t h e S1 sit e, t h e S1ʹ′ a n d S2ʹ′ sit es ca n be
r edesign ed ea sily. A sin gle su bst it u t ion , Lys60Glu ,
is su fficien t t o cr ea t e a t r ypsin va r ia n t wit h a ∼100fold pr efer en ce for Ar g a t S1ʹ′.229 Th e con st r u ct ion of
a m et a l bin din g sit e a t S2ʹ′ a s descr ibed a bove wa s
su fficien t t o cr ea t e specificit y for P 2 H is.223 Wh y a r e
t h e S1 sit es of t r ypsin a n d ch ym ot r ypsin so difficu lt
t o r edesign , especia lly wh en com pa r ed wit h t h e S1ʹ′
sit e? P a r t of t h e a n swer is u n dou bt edly in t opology
of t h e sit es. Th e S1 sit e is a deep pocket wh ich
su r r ou n ds t h e P 1 r esidu e. A ca vit y is a n in t r in sica lly
u n st a ble st r u ct u r e a n d less a ble t o t oler a t e su bst it u t ion s. In con t r a st , t h e S1ʹ′ sit e is a sh a llow gr oove.
Mor eover , t h e S1 sit e of t r ypsin is pa r t of t h e
a ct iva t ion dom a in of t r ypsin . Th e S1 sit e h a s t wo
con for m a t ion s, t h e or der ed st r u ct u r e of t h e m a t u r e
pr ot ea se a n d a n in a ct ive, zym ogen -like con for m a t ion .
It is n ot su r pr isin g t h a t t h e Asp189Ser m u t a t ion
ca u ses com plet e defor m a t ion of t h e S1 sit e, pu sh in g
t h e con for m a t ion a l equ ilibr iu m t o t h e zym ogen -like
for m .221,230 In effect , t h e S1 sit e is “bu ilt t o colla pse”.
In con t r a st , t h e st r u ct u r e of t h e S1ʹ′ sit e r em a in s
con st a n t du r in g zym ogen a ct iva t ion , a n d is in h er en t ly m or e st r u ct u r a lly sou n d t h a n t h e S1 sit e. Th e
st a bilit y of t h e fr a m ewor k is obviou sly a n im por t a n t
det er m in a n t of t h e a bilit y t o r edesign a n en zym e.
A well-or der ed S1 sit e m a y n ot be a n a bsolu t e
r equ ir em en t for h igh pr ot ea se a ct ivit y, so lon g a s t h e
con ver sion t o a ca t a lyt ica lly com pet en t st r u ct u r e
does n ot in voke a h u ge en er get ic pen a lt y. Tr f
Ch [S1+L1+L2] is a lm ost a s a ct ive a s ch ym ot r ypsin
a lt h ou gh it s S1 sit e is defor m ed. H owever , t h e r igid
S1 sit e of ch ym ot r ypsin is a lm ost cer t a in ly a r equ ir em en t for su r viva l in it s ph ysiologica l n ich est h e
pr ot ea se-r ich du oden u m . It is wor t h r em em ber in g
t h a t en zym es evolve t o opt im ize a ll a spect s of t h eir
ph ysiologica l fu n ct ion s, in clu din g ca t a lysis, expr ession , t r a n sla t ion , foldin g, a n d degr a da t ion .
B. Redesigning Esterase Activity
In con t r a st t o a m ide h ydr olysis, t h e est er a se specificit y of t h e S1 sit e ca n be ea sily r e-en gin eer ed. Th e
Asp189Ser m u t a t ion is su fficien t t o ch a n ge t h e
specificit y of t r ypsin t o t h a t of ch ym ot r ypsin in est er
h ydr olysis.200 Tr ypsin h a s been con ver t ed in t o a n
ela st a se-like est er a se, bu t t h is en zym e h a s n o m ea s-
Chemical Reviews, 2002, Vol. 102, No. 12 4517
u r a ble a m ida se a ct ivit y.227 Sim ila r ly, t h e pr efer en ce
of gr a n zym e B for a cidic P 1 r esidu es ca n be ch a n ged
t o P h e wit h t h e su bst it u t ion of Ar g226 wit h Gly
(u n for t u n a t ely, t h is wor k did n ot u t ilize pu r ified
en zym es, a n d t h er efor e m u st be in clu ded wit h ca u t ion ).231 Th e ea se wit h wh ich est er a se a ct ivit y ca n be
m a n ipu la t ed der ives fr om t h e m u ch gr ea t er in t r in sic
r ea ct ivit y of est er s a s discu ssed a bove.
VII. How Do Remote Interactions Translate into
Catalysis?
Th e oper a t ion of t h e ca t a lyt ic m a ch in er y in t h e
im m edia t e vicin it y of t h e scissile bon d a ppea r s ver y
st r a igh t for wa r d: Asp102, H is57, a n d Ser 195 a n d t h e
oxya n ion h ole a r e a lign ed t o fa cilit a t e pr ot on t r a n sfer s a n d st a bilize t h e ch a r ges t h a t develop a s t h e
r ea ct ion pr oceeds. It is a lso clea r t h a t a n et wor k of
h ydr ogen bon ds pr opa ga t es ou t wa r d fr om t h e ca t a lyt ic t r ia d, so t h a t ch a n ges in bon din g or der a n d
charge distribution at the scissile bond will propagate
t o m or e r em ot e en zym e-su bst r a t e in t er a ct ion s, a n d
vice ver sa . In t er a ct ion s fa r fr om t h e scissile bon d, in
t h e S1 sit e, polypept ide sit e, S2-Sn or S1ʹ′-Sn ʹ′, ca n
dr a m a t ica lly a n d cooper a t ively a cceler a t e t h e h ydrolysis of substrate. Such interactions must optimize
t h e fu n ct ion of t h e ca t a lyt ic m a ch in er y.
Th e in flu en ce of r em ot e bin din g in t er a ct ion s on t h e
catalytic triad is illustrated by trifluoroketone inhibit ion . Th e H is57 Nδ1 pr ot on s h a ve low-field ch em ica l
sh ift s, eleva t ed pK a ’s, low fr a ct ion a t ion fa ct or s a n d
slow r a t es of D 2 O exch a n ge.149,150,165 F or a ser ies of
in h ibit or s va r yin g in t h e P 2 r esidu e, t h ese pr oper t ies
cor r ela t e wit h t h e a ffin it y of t h e in h ibit or a s well a s
k ca t /K m for t h e a n a logou s su bst r a t e.150,151,163,165,232 Of
cou r se, t h e big qu est ion is h ow do t h ese r em ot e
in t er a ct ion s t r a n sla t e in t o ca t a lysis. Most r em ot e
in t er a ct ion s do n ot in cr ea se su bst r a t e a ffin it y bu t
con t r ibu t e exclu sively t o ca t a lysis. Th is obser va t ion
su ggest s t h a t t h ese in t er a ct ion s in t r odu ce st r a in in t o
t h e su bst r a t e, wh ich pen a lizes bin din g a ffin it y bu t
a cceler a t es ca t a lysis. Th is st r a in ca n t a ke m a n y
forms, including desolvation of enzyme and substrate,
lockin g t h e su bst r a t e in t h e ca n on ica l con for m a t ion ,
in t er a ct ion s of t h e ca r bon yl wit h t h e oxya n ion h ole,
da m pin g of pr ot ein m ot ion s, con for m a t ion a l ch a n ges
a n d/or geom et r ic dist or t ion . Wh a t follows a r e som e
possible m ech a n ism s, in n o pa r t icu la r or der . Th ese
m ech a n ism s a r e n ot m u t u a lly exclu sive, a n d it is
likely t h a t sever a l a r e oper a t in g.
A. Remote Interactions Align the Substrate in the
Active Site
On e pr oposa l is t h a t r em ot e bin din g in t er a ct ion s
a lign t h e su bst r a t e r ela t ive t o t h e ca t a lyt ic t r ia d,
pla cin g t h e su bst r a t e in ca t a lyt ic r egist er .15 Th is
m ech a n ism is pa r t icu la r ly a t t r a ct ive wh en con sider in g t h e pr ot ea se su bsit es im m edia t ely a dja cen t t o t h e
scissile bon d, i.e., S2, S1, a n d S1ʹ′. Th ese in t er a ct ion s
dock t h e scissile bon d n ext t o Ser 195 a n d t h e oxya n ion h ole. Un for t u n a t ely, t h e a lign m en t m ech a n ism
becom es less a ttra ctive a s th e in tera ction s get fa r t her
fr om t h e sit e of ch em ica l t r a n sfor m a t ion . On ce t h e
P 1/S1 a n d P 2/S2 in t er a ct ion s a r e for m ed, t h e su b-
4518 Chemical Reviews, 2002, Vol. 102, No. 12
Hedstrom
st r a t e is t et h er ed in posit ion ; t h er e wou ld seem t o
be lit t le ga in ed for in t er a ct ion s beyon d t h e S2 sit e.
An ot h er m ech a n ism wou ld a ppea r n ecessa r y t o h a r n ess t h e bin din g en er gy a t t h ese m or e r em ot e sit es.
An y expla n a t ion for h ow dist a l in t er a ct ion s a cceler a t e pr ot ea se ca t a lysis m u st a lso expla in t h e h ow
t h ese in t er a ct ion s ca n a ct “in t r a n s”. F or exa m ple,
a m in es or gu a n idin iu m com pou n ds a cceler a t e t h e
h ydr olysis of su bst r a t es con t a in in g P 1-Gly or Ala by
t r ypsin .233,234 Th is obser va t ion su ggest s t h a t t h e P 1S1 in t er a ct ion does n ot sim ply dock t h e su bst r a t e in
the a ctive site cleft. E thyla mine could trigger a su btle
con for m a t ion a l ch a n ge t h a t a ct iva t es t h e en zym e.
Th e P 1/S1 in t er a ct ion m a y pr even t n on pr odu ct ive
bin din g m odes, t h u s in dir ect ly im pr ovin g t h e a lign m en t of t h e su bst r a t e in t h e a ct ive sit e. H owever , t h e
a lign m en t m ech a n ism does n ot a ppea r t o be su fficien t t o expla in t h e effect s of t h e r em ot e bin din g
in t er a ct ion s on ca t a lysis.
B. Remote Interactions Are Optimized in the
Transition State
E n zym es a r e oft en descr ibed a s t em pla t es for t h e
t r a n sit ion st a t e of a r ea ct ion . Con ven t ion a l views of
t h e t r a n sit ion st a t e of pr ot ea se r ea ct ion s focu s on t h e
sit e of bon d m a kin g/br ea kin g a n d for m a t ion of t h e
t et r a h edr a l a ddu ct . H owever , if r em ot e in t er a ct ion s
increase catalysis, then remote interactions must also
be pa r t of t h e t r a n sit ion st a t e. Th er efor e, a com plet e
descr ipt ion of t h e t r a n sit ion st a t e m u st in clu de t h e
P /S a n d P ʹ′/Sʹ′ in t er a ct ion s in a ddit ion t o t h e t et r a h edr a l a ddu ct , ca t a lyt ic t r ia d a n d oxya n ion h ole. Th e
m ost dr a m a t ic ch a n ges in st r u ct u r e a r e in volved in
t h e bon d m a kin g a n d br ea kin g a t t h e scissile bon d,
wh ile sm a ller ch a n ges r esu lt fr om t h e ch a n ges in
geom et r y in t h e t r a n sfor m a t ion fr om pla n a r su bst r a t e t o t et r a h edr a l t r a n sit ion s st a t e. Th ese sm a ll
ch a n ges ca n pr opa ga t e in t o sign ifica n t displa cem en t s
down t h e pept ide ch a in , opt im izin g t h e r em ot e bin din g sit e in t er a ct ion s in t h e t r a n sit ion st a t e.235 Su ch
ch a n ges cou ld pr ovide a m ech a n ism for t h e r a t e
a cceler a t ion . Su ppor t for t h is m ech a n ism ca n be
fou n d in t h e in h ibit ion of ela st a se by pept idyl a ldeh ydes.47 Wh ile t h e P 4 r esidu e in cr ea ses t h e a ffin it y
of a n a lcoh ol in h ibit or by 10-fold, t h e a ffin it y of t h e
cor r espon din g a ldeh yde in h ibit or in cr ea ses by 100fold (Table 7). This observation demonstrates that the
P 4-S4 in t er a ct ion is opt im ized in a t et r a h edr a l
a ddu ct . Sim ila r ly, t h e h ydr ogen bon d bet ween t h e
P 1-NH a n d t h e Ser 214 ca r bon yl a ppea r s t o be
st r on ger in t r a n sit ion st a t e a n a logu e com plexes t h a n
in pr ot ein in h ibit or com plexes (Ta ble 8).45,236 Th ese
obser va t ion s su ggest t h a t t h e in t er a ct ion s a t t h e
r em ot e sit es a r e opt im ized in t h e t r a n sit ion st a t e.
Ta ble 7. In h ibitio n o f P o rc in e P a n c re a tic Ela s ta s e by
P e p tid y l Ald e h y d e s a
a
in h ibit or
K i (µM)
Ac-Ala -P r o-Ala -CH 2 OH
Ac-P r o-Ala -P r o-Ala -CH 2 OH
Ac-Ala -P r o-Ala -CH O
Ac-P r o-Ala -P r o-Ala -CH O
7000
600
62
0.8
Da t a fr om r ef 47.
Ta ble 8. Op tim iza tio n o f Hy d ro g e n B o n d s in
Tra n s itio n S ta te Co m p le x e s a
h ydr ogen bon d
bor oVa l
Ser 195 N-P 1 O1
Gly193 N-P 1 O1
H is57 N!2-P 1 O2
Ser 214 O-P 1 N
Gly216 N-Ala P 3 O
Gly216 O-Ala P 3N
H is57 N!2-Ser 195 Oγ
Leu 41 O-P 2ʹ′ N
Leu 41 N-P 2ʹ′ O
2.9
2.6
2.7
3.0
3.0
2.9
3.0
P va l-La c
(P va l)
2.8
2.6
5.5 (2.9)b
3.0
3.0
2.9
7.1 (3.0)
2.8
3.0
ovom u coid
3.1
2.6
3.6
2.9
3.0
2.6
2.9
3.1
a
X-r a y cr yst a l st r u ct u r es of a lph a lyt ic pr ot ea se com plexes
wit h Boc-Ala -P r o-bor o-Va l, 37 Boc-Ala -Ala -P r o-P va lLa c-Ala , wh er e P va l is t h e ph osph on ic a cid a n a logu e of Va l
a n d La c is la ct a t e 45 a n d Boc-Ala -Ala -P r o-P va l (in pa r en t h eses) a n d ovom u coid t h ir d dom a in 236 a r e com pa r ed. Da t a
from ref 45. b In the Boc-Ala-Ala-Pro-Pval-Lac-Ala, His57
h a s r ot a t ed a wa y fr om t h e in h ibit or , pr esu m a bly du e t o t h e
a bsen ce of a n ega t ive ch a r ge in t h e n eu t r a l ph osph on a t e. Th e
N!1 h ydr ogen bon d is pr esen t in t h e ch a r ged ph osph on a t e.
C. Remote Interactions Induce a Conformational
Change that Favors Catalysis
On e oft -in voked m ech a n ism for u t ilizin g r em ot e
bin din g in t er a ct ion s is in du ced fit , i.e., r em ot e in t er a ct ion s in du ce a con for m a t ion a l ch a n ge t h a t a lign s
t h e ca t a lyt ic m a ch in er y, a ct iva t in g t h e en zym e.
However, n o con form a tion a l ch a n ge is obser ved when
chymotrypsin or trypsin bind substrates or inhibitors,
a n d t h e a lign m en t of t h e ca t a lyt ic t r ia d does n ot
ch a n ge. Th u s, a cla ssica l in du ced fit m ech a n ism is
n ot oper a t ive a n d ser in e pr ot ea ses a ppr oa ch t h e idea l
of t h e en zym e a s a t em pla t e for t h e t r a n sit ion st a t e
(exa m ples of ser in e pr ot ea ses t h a t do u t ilize a n
in du ced fit m ech a n ism will be discu ssed below).
However, subtle conformational changes are observed
wh en a poen zym e a n d in h ibit or com plexes a r e ca r efu lly com pa r ed. F or exa m ple, wh en t r ypsin bin ds a
peptidyl boronic a cid, sma ll a djustments a re observed
in ∼50 of t h e 224 r esidu es,237 a n d sim ila r a dju st m en t s a r e a lso obser ved in bor on ic a cid st r u ct u r es
of alpha lytic protease.37 These structural changes are
sm a ll a n d seem u n likely t o in volve a sign ifica n t
en er get ic ba r r ier . Ra t h er , t h is st r u ct u r a l pla st icit y
is pr oba bly r equ ir ed t o a ccom m oda t e pr odu ct a ssocia t ion /dissocia t ion a n d t h e differ en t t r a n sit ion
st a t es of t h e pr ot ea se r ea ct ion .
If good su bst r a t es in du ce con for m a t ion a l ch a n ges
t h a t in cr ea se t h e r ea ct ivit y of t h e ca t a lyt ic t r ia d a n d
oxya n ion h ole, t h en disr u pt ion s of t h e ca t a lyt ic
m a ch in er y sh ou ld h a ve a m or e delet er iou s effect on
t h e h ydr olysis of good su bst r a t es. Un for t u n a t ely,
t h er e a r e lit t le da t a on t h is poin t beca u se t h ese
m u t a t ion s decr ea se a ct ivit y t o t h e poin t wh er e on ly
good su bst r a t es ca n be m on it or ed. Th e su bst it u t ion
of Asp102 wit h Asn a ppea r s t o h a ve a m or e delet er ious effect on the hydrolysis of peptide substrates than
ot h er su bst r a t es, bu t t h is differ en ce is n ot obser ved
wh en t h e pH -in depen den t r ea ct ion is con sider ed.79
Mu t a t ion s of H is57 do ch a n ge specificit y, bu t t h is is
expect ed beca u se H is57 for m s pa r t of bot h t h e S2 a n d
S1ʹ′ sit es (F igu r es 7). Un for t u n a t ely, it will be difficu lt
t o cor r ela t e su ch su bt le st r u ct u r a l differ en ces t o
ca t a lysis.
Serine Protease Mechanism and Specificity
D. The Remote Interactions Shield the Catalytic
Triad from Solvent
Ma n y en zym es, even pr ot ea ses, h a ve m ot ile fla ps
t h a t close over t h e a ct ive sit es, pr even t in g solven t
a ccess t o t h e a ct ive sit e. Su ch desolva t ion pr ovides a
sim ple m ech a n ism t o a ch ieve la r ge r a t e a cceler a t ion s.262,264 Th e r em ot e por t ion s of t h e pept ide su bst r a t e cou ld per for m t h is t a sk for ser in e pr ot ea ses.
In pa r t icu la r , t h e P 2 a n d P 1ʹ′ r esidu es will sh ield
His57 from bulk solvent. This effect ca n be illustrated
by t h e exch a n ge of t h e pr ot on s of H is57 wit h solven t
in t r iflu or oket on e com plexes. Th e r a t e of exch a n ge
decreases markedly with better P2 residues (from 280
t o 12 s -1 ; t h ese r a t es a r e 100× gr ea t er t h a n t h e r a t e
of in h ibit or dissocia t ion , in dica t in g t h a t pr ot on exch a n ge depen ds on t h e diffu sion of wa t er in t o t h e
com plex).150 Th u s, t h e P 2 r esidu e blocks wa t er a ccess
t o t h e ca t a lyt ic t r ia d. In t er a ct ion s a t m or e r em ot e
sit es cou ld sim ila r ly r est r ict solven t a ccess a s well
a s lower t h e dielect r ic con st a n t of t h e a ct ive sit e.
Similarly, the “trans” phenomenon could be explained
by t h e expu lsion of wa t er fr om t h e a ct ive sit e, or
perhaps by neutralizing the negative charge of Asp189,
fa vor in g ca t a lysis by lower in g t h e dielect r ic con st a n t
in t h e vicin it y of t h e scissile bon d. Not e t h a t t h e t igh t
pa ckin g of en zym e a n d su bst r a t e ca n ca u se st er ic
com pr ession t h a t will a lso fa cilit a t e pr ot on t r a n sfer .23,163,238
Chemical Reviews, 2002, Vol. 102, No. 12 4519
VIII. Specificity via Induced Fit and Allostery
Th e a bove discu ssion focu sed on specificit y t h a t
der ived fr om a r ela t ively r igid a ct ive sit e cleft a s
fou n d in ch ym ot r ypsin , t r ypsin , a n d ela st a se. Ser in e
pr ot ea se specificit y h a s a lso been a scr ibed t o cla ssica l
in du ced fit m ech a n ism s a n d a llost er ic in t er a ct ion s.
Th e followin g will pr ovide exa m ples of t h ese ph en om en a wit h com plem en t pr ot ea se F a ct or D, t h e
ba ct er ia l en zym e R-lyt ic pr ot ea se a n d t h e coa gu la t ion
pr ot ea se t h r om bin .
A. Induced Fit as a Mechanism for Narrow
Specificity
It is im por t a n t t o r ecogn ize t h a t a n effect ive
induced fit mecha nism requires more tha n the simply
pla cin g a con for m a t ion a l ch a n ge in t h e r ea ct ion
coor din a t e (F igu r e 15).240,241 An en zym e t h a t ca n
r a pidly equ ilibr a t e bet ween in a ct ive a n d a ct ive con for m a t ion s will be n o m or e specific t h a n a n en zym e
t h a t does n ot u n der go a con for m a t ion a l ch a n gest h e
equilibrium between inactive and active forms simply
lower s t h e fr a ct ion of a ct ive en zym e. Th is m u st be
t h e ca se if t h e ch em ica l t r a n sfor m a t ion is r a t elim it in g for t h e r ea ct ion ; t h e su bst r a t e bin din g st eps
will be in equ ilibr iu m , a n d n o a dva n t a ge is ga in ed
fr om t h e con for m a t ion a l ch a n ge.
E. Remote Interactions Couple Catalysis to
Motion of the Protease Structure
P r ot ein dyn a m ics is t h e wild ca r d in pr ot ea se
catalysis and specificity, as is true of enzyme catalysis
a s a wh ole. Th e idea t h a t r ea ct ion dyn a m ics a r e
cou pled t o pr ot ein m ot ion s h a s lon g in t r igu ed bioch em ist s. Su ch m ot ion s m igh t be u sed t o m ech a n ica lly st r ess t h e pept ide bon d, disr u pt in g r eson a n ce
st a biliza t ion a n d a ct iva t in g t h e ca r bon yl for n u cleoph ilic a t t a ck.239 Likewise, t h e a t t a ck of Ser 195 on t h e
scissile bon d m igh t be lin ked t o m ovem en t of t h e
su r r ou n din g r esidu es. Less dr a m a t ica lly, dyn a m ic
m ovem en t cou ld sim ply be im por t a n t for su bst r a t e
a ssocia t ion a n d pr odu ct dissocia t ion . Th e loca t ion of
t h e a ct ive sit e bet ween t h e t wo bet a ba r r els pla ces
t h e su bst r a t e a t ju n ct ion of dom a in m ot ion s in
ch ym ot r ypsin -like pr ot ea ses.
Th e ser in e pr ot ea ses pr ovide a u n iqu e oppor t u n it y
t o a ddr ess t h is qu est ion : if su ch pr ot ein m ot ion is
r equ ir ed for ca t a lysis, t h en sim ila r m ot ion s sh ou ld
be obser ved in ch ym ot r ypsin -like a n d su bt ilisin -like
pr ot ea ses, a s well a s t h e ot h er ser in e pr ot ea se
fa m ilies. On t h e ot h er h a n d, it is difficu lt t o see h ow
pr ot ein dyn a m ics cou ld be m a in t a in ed in fou r differ en t st r u ct u r es. It m a y be possible t o m on it or pr ot ein
m ot ion s du r in g ser in e pr ot ea se ca t a lysis. Su ch exper im en t s best u t ilize r ever sible r ea ct ion s, wh ich h a s
t h wa r t ed t h eir a pplica t ion t o pr ot ea se r ea ct ion s.
H owever , pr ot ea ses a lso ca t a lyze t h e exch a n ge of
wa t er in t o t h e C-t er m in u s of a pr odu ct ca r boxylic
a cid. Th is r ea ct ion cou ld be exploit ed t o in vest iga t e
pr ot ea se m ot ion s du r in g ca t a lysis.
F ig u re 15. Th e effect of a con for m a t ion a l ch a n ge on
specificit y. Sim ply a ddin g a con for m a t ion a l ch a n ge t o a n
en zym e does n ot pr ovide specificit y r ela t ive t o a n en zym e
t h a t does n ot u n der go a con for m a t ion a l ch a n ge. As sh own
in Sch em es 1 a n d 2, t h e con for m a t ion a l ch a n ge sim ply
decr ea ses t h e a m ou n t of a ct ive en zym e for m . H owever ,
wit h t h e a dded con st r a in t t h a t t h e con for m a t ion a l ch a n ge
is r a t e-lim it in g, a n d t h e good su bst r a t e a cceler a t es t h e
con for m a t ion a l ch a n ge, specificit y ca n be a ch ieved beca u se
t h e bin din g st eps a r e n o lon ger a t equ ilibr iu m . If bot h
con for m a t ion s a r e a ct ive a s in Sch em e 3, br oa d specificit y
ca n be a t t a in ed.
4520 Chemical Reviews, 2002, Vol. 102, No. 12
Th is pr in ciple is illu st r a t ed in t h e t r ypsin /t r ypsin ogen syst em , wh er e m u t a t ion s t h a t a lt er t h e
equ ilibr iu m bet ween a ct ive a n d in a ct ive con for m a t ion s do n ot ch a n ge specificit y.242 Th e Ile16-Asp194
sa lt br idge ca n be dest a bilized by t h e su bst it u t ion of
Ile16 wit h sm a ller r esidu es, fa vor in g t h e in a ct ive
zym ogen -like con for m a t ion . Su ch m u t a t ion s decr ea se
t h e h ydr olysis of good a n d poor su bst r a t es equ iva len t ly, in clu din g bot h a m ides a n d est er s.243 In a ddit ion , m u t a t ion s ca n be in t r odu ced t h a t a ct iva t e
t r ypsin ogen by st a bilizin g t h e m a t u r e pr ot ea se con for m a t ion .244 Th e equ ilibr iu m bet ween in a ct ive a n d
a ct ive con for m a t ion s ca n be det er m in ed by m ea su r in g t h e a ffin it y of BP TI, wh ich in du ces t h e m a t u r e
pr ot ea se con for m a t ion .242 Th e decr ea se in va lu es of
k ca t /K m cor r ela t es wit h t h e decr ea se in a ffin it y of
BP TI, a s expect ed for t h e m ech a n ism of F igu r e 15.
An in du ced fit m ech a n ism ca n im pa r t specificit y
wh en t h e con for m a t ion a l ch a n ge is r a t e-lim it in g. In
t h is ca se, a good su bst r a t e ca n a cceler a t e t h e r a t e of
t h e con for m a t ion a l ch a n ge, a n d t h u s a cceler a t e t h e
r ea ct ion . Th e bin din g st eps will n ot be a t equ ilibr iu m
beca u se t h e su bst r a t e will r a pidly r ea ct t o pr odu ct s.
F u r t h er discr im in a t ion ca n be a ch ieved if ch em ist r y
is slow for t h e poor su bst r a t e.241
B. Factor D: An Induced Fit Protease
Su ch a n in du ced fit m ech a n ism ca n a ccou n t for t h e
r em a r ka ble specificit y of F a ct or D. Un like m ost
ser in e pr ot ea ses, F a ct or D cir cu la t es in pla sm a in it s
m a t u r e for m , a n d t h er efor e m u st h a ve ext r a or din a r y
specificit y. F a ct or D h a s on ly on e n a t u r a l su bst r a t e,
t h e com plem en t com plex C3bB.245 Rem a r ka bly, k ca t /
K m for h ydr olysis of C3bB is 10 6 M -1 s -1 , com pa r a ble
t o t h e h ydr olysis of a good pept ide su bst r a t e by
trypsin. However, Fa ctor D ha s little estera se activity
(k ca t /K m ) ∼300 M -1 s -1 com pa r ed t o 10 7 M -1 s -1 for
t r ypsin 197 ) a n d ca n n ot h ydr olyze pept ides con t a in in g
t h e C3bB clea va ge sit e.197 Th e st r u ct u r e of F a ct or D
does n ot r esem ble a n a ct ive ser in e pr ot ea se, wh ich
expla in s t h ese pu zzlin g obser va t ion s.246 Th e S1 sit e,
S2-S4 sit es a n d ca t a lyt ic t r ia d a r e defor m ed du e t o
t h e pr esen ce of Ser 94, Th r 214, a n d Ser 215 a t posit ion s u su a lly occu pied by Tr p/Tyr , Ser , a n d Tr p/P h e,
r espect ively. Su bst it u t ion of t h ese r esidu es r et u r n s
t h e ca t a lyt ic t r ia d t o a n or m a l con for m a t ion , bu t on ly
in cr ea ses t h e est er a se a ct ivit y 10×.247 Th e bin din g
of C3bB m u st r epa ir t h ese defect s a n d in du ce F a ct or
D t o a ssu m e t h e u su a l a ct ive con for m a t ion .
C. Alpha Lytic Protease: Induced Fit as a
Mechanism for Broad Specificity
In du ced fit a lso pr ovides a m ech a n ism for expa n din g specificit y.248 Im a gin e a n en zym e h a s sever a l
con for m a t ion s of com pa r a ble en er gies, ea ch of wh ich
a ccom m oda t es a differ en t su bst r a t e or , m or e pr ecisely, t h e t r a n sit ion st a t e for a differ en t su bst r a t e.
Each substrate will induce the appropriate conformat ion for ca t a lysis, a n d t h e en zym e ca n h ydr olyze
sever a l su bst r a t es wit h com pa r a ble va lu es of k ca t /K m .
Su ch a n in du ced fit m ech a n ism pr ovides t h e st r u ct u r a l pla st icit y n ecessa r y for br oa d specificit y.
Hedstrom
Ta ble 9. S p e c ific ity o f Wild Ty p e a n d M192A Alp h a
Ly tic P ro te a s e a
k ca t /K m M -1 s -1
su bst r a t e
Su c-Ala -Ala -P r o-Ala -pNA
Su c-Ala -Ala -P r o-Va l-pNA
Su c-Ala -Ala -P r o-Met -pNA
Su c-Ala -Ala -P r o-Leu -pNA
Su c-Ala -Ala -P r o-P h e-pNA
a
wild t ype
2.1 ×
790
1800
4.1
0.38
10 4
M192A
1 × 10 4
3 × 10 3
3.5 × 10 5
1.1 × 10 5
3.1 × 10 5
Da t a fr om r ef 249.
Th e br oa d specificit y of t h e Met 192Ala m u t a n t of
a lph a lyt ic pr ot ea se ca n be u n der st ood in t er m s of
t h is st r u ct u r a l pla st icit y (a lt h ou gh in du ced fit is
pr oba bly n ot a n a ppr opr ia t e t er m sin ce t h e ca t a lyt ic
m a ch in er y is a lwa ys a lign ed). Alph a lyt ic pr ot ea se
pr efer s su bst r a t es wit h sm a ll a liph a t ic r esidu es a t
P 1. Th e S1 sit e is sm a ll a n d r ela t ively r igid, occlu ded
by Met 192. Su bst it u t ion of Met 192 wit h Ala wou ld
be expect ed t o cr ea t e a la r ge h ydr oph obic S1 sit e
sim ila r t o ch ym ot r ypsin a n d t h e X-r a y cr yst a l st r u ct u r e of t h e u n liga n ded m u t a n t pr ot ea se sh ows t h a t
t h e S1 sit e is en la r ged. H owever , u n like ch ym ot r ypsin , Met 192Ala h ydr olyzes P 1-Ala a n d P h e
su bst r a t es wit h com pa r a ble va lu es of k ca t /K m (Ta ble
9).249 In h ibit or com plexes sh ow t h a t t h e st r u ct u r e of
t h e S1 sit e of Met 192Ala is n ot a sim ple h ydr oph obic
pocket , bu t a da pt s t o a ccom m oda t e differ en t P 1
r esidu es.251 Th er efor e, t h e S1 sit e displa ys st r u ct u r a l
pla st icit y wh er ein ea ch su bst r a t e in du ces t h e a ppr opr ia t e con for m a t ion for h ydr olysis. Th e m u t a t ion
a ppea r s t o h a ve a pr ofou n d effect on t h e dyn a m ic
pr oper t ies of t h e S1 sit e. In wild-t ype a lph a lyt ic
pr ot ea se, t h e wa lls of t h e S1 sit e a r e cou pled a n d
m ove t oget h er a s a u n it so t h a t t h e dim en sion s of
t h e S1 sit e a r e m a in t a in ed.252 H owever , t h e Met 192
m u t a t ion disr u pt s t h is cou plin g, a llowin g t h e t wo
wa lls of t h e S1 sit e t o m ove in depen den t ly a n d t h e
dim en sion s of t h e S1 sit e t o va r y. 250,253,254 Th u s, t h e
m u t a n t en zym e ca n sa m ple m a n y differ en t con for m a t ion s of t h e S1 sit e, a n d h ydr olyze m a n y differ en t
su bst r a t es.
Th r om bin pr ovides a secon d exa m ple of h ow specificit y ca n be expa n ded by t h e a bilit y t o a ccess
m u lt iple con for m a t ion s. Th r om bin ’s r ole in t h e for m a t ion of a blood clot is well-kn own . 265,266 Less
a ppr ecia t ed is t h r om bin ’s a n t icoa gu la t ion fu n ct ion .
Th e su bst r a t es of t h e coa gu la t ion pa t h wa y (fibr in ogen , fa ct or V, fa ct or VIII, a n d fa ct or XIII) con t a in
n eu t r a l or posit ively ch a r ged P 3 a n d P 3ʹ′ r esidu es,
wh ile t h e su bst r a t es of t h e a n t icoa gu la t ion pa t h wa y
(e.g., pr ot ein C) con t a in Asp a t t h ese posit ion s.
Th rom bin requ ires two con forma tion s to process such
differ en t su bt r a t es.255,256 Th e pr ocoa gu la n t con for m a t ion , or “fa st ” for m , h ydr olyzes fibr in ogen ∼10 3 t im es
fa st er t h a n pr ot ein C. In con t r a st , t h e “slow” for m
h a s a 50-fold pr efer en ce for pr ot ein C. 255 Im por t a n t ly,
Na + is a n a llost er ic effect or of t h is con for m a t ion a l
switch, binding specifica lly to the fa st form at a single
sit e.257 Th e fa st a n d slow for m s a r e a ppr oxim a t ely
equ a l a t ph ysiologica l Na + con cen t r a t ion s. Na + is a n
a llost er ic effect or of sever a l ot h er ser in e pr ot ea ses,
in clu din g F a ct or Xa , a ct iva t ed pr ot ein C, a n d F a ct or
IXa ; Na + r egu la t ion cor r ela t es wit h t h e pr esen ce of
Serine Protease Mechanism and Specificity
Tyr or P h e a t posit ion 225, wh ile Na + in depen den ce
cor r ela t es wit h P r o225.258 Th e Na + bin din g sit e lies
bet ween t h e 180 a n d 220 loops.259 Su bst it u t ion of
Tr p215 a n d Glu 217 a br oga t es Na + bin din g, st a bilizin g t h e slow for m wh ile Glu 39, Tr p60d, Glu 192,
Asp221, a n d Asp222 for m a n “a llost er ic cor e” t h a t
con n ect s t h e Na + bin din g sit e t o t h e a ct ive sit e cleft .
IX. How Serine Proteases Work: Summary and
Prospects
Ser in e pr ot ea ses a r e pr oba bly t h e m ost t h or ou gh ly
in vest iga t ed en zym e syst em . Wh ile t h e fu n ct ion of
t h e ca t a lyt ic t r ia d a n d oxya n ion h ole ca n be r a t ion a lized in t er m s of elect r ost a t ic st a biliza t ion of ch a r ges
developin g in t h e t r a n sit ion st a t e, h ow r em ot e bin din g in t er a ct ion s fa cilit a t e ca t a lysis r em a in s a m yst er y. Ca t a lysis a n d specificit y a r e n ot sim ply con t r olled by a few r esidu es, bu t a r e r a t h er a pr oper t y
of t h e en t ir e pr ot ein fr a m ewor k, con t r olled via t h e
dist r ibu t ion of ch a r ge a cr oss a n et wor k of h ydr ogen
bon ds a n d per h a ps a lso by t h e cou plin g of dom a in
m ot ion t o t h e ch em ica l t r a n sfor m a t ion . Ser in e pr ot ea ses pr esen t a u n iqu e oppor t u n it y t o t est t h ese
idea s.
X. Useful Web Sites
Rose, T., a n d Di Cer a , E . Ser in e P r ot ea se H om e
P a ge.
h t t p://www.bioch em .wu st l.edu /∼pr ot ea se/
in dex.h t m l.
Ra wlin gs, N. D., O’Br ien , E . A., a n d Ba r r et t , A. J .
2002 ME ROP S: Th e P r ot ea se Da t a ba se. N u cleic
Acid s R es. 30, 343. h t t p://m er ops.ia pc.bbsr c.a c.u k/.
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