R e p r i n t e d f r o m t h e J o u r n a l o f t h e A m e r i c a n C h e m i c a l S o c i e t y , 1 9 E 5 ,I 0 2 , 6 9 9 9 .
Copyright O 1985by the American Chemical Society and reprinted by permission of the copyright owner.
Enzyme-Catalyzed
OrganicSynthesis:A Comparison
of
for in Situ Regeneration
Strategies
of NAD from NADH
Linda G. L€e and George M. Whitesides*
Contributionfrom the Departmentof Chemistry,Haru-ardUniuersity,
Cambridge,Massachusetts02138,and Massachusetts
Institute of Technology,
Cambridge,Massachusetts
02139. Receiued
February25, 1985
Abstnct: This papercomparesthreedifferenttypesof enzvmaticsvstemsfor in situ regeneration
of NAD from NADH for
usein practical-scale
enzymc-catalyzed
organicsynthesis.The first. and the mostgenerallyuseful.usesan organicoxidant
quantities(2-oxoglutarate,
in stoichiometric
with catalysisby glutamatedehydrogenase);
the secondinvolvesdioxygenas the
terminaloxidant.with an intermediateelectroncarrierdye (methylcneblue,with a diaphorase
as catalyst):the third is based
on a stoichiometric
inorganicoxidant(fenicyanide,with diaphorase
as catalyst).The relativementsof theseand other NAD
regeneration
systemsare discussed
with particularreferenceto intrinsickinetic and thermodynamiclimitationsto practical
application. The pap€r includes representativeexamplesof oxidationsusing each regenerationsystem. For 2-oxoglutantef glutamatedehydrogenase
and methyleneblue/diaporase/O2,the conversionof cis-cyclohexanedimethanol
to
(+)-(lR,6.5)'cis-8-oxabicyclo[4.3.0]nonan-7-one
catalyzedby horseliveralcoholdehydrogenase
wascarriedout on 70- and
30-mmolscales.respcctively.The lessusefulferricyanide/diaphorase
systemwas testedon a S-mmolscalein the oxidation
of glucse to gluconatecatalyzedby glucoscdehydrogenasc.For many dehydrogenas€-catalyzed
oxidations,the most important
limitationsto syntheticapplicationseemto lie not in NAD regeneration
but in the unrelatedproblemof noncompetitive
inhibition
by product. The paperdescribesempincal relationship betweenthe equilibrium constantsfor the oxidationor reductionreactions
beingconsidered
and the valuesof Michaelisand productinhibitionconstants.Theserelationships
are usefulin identifying
reactionswhich are plausiblecandidatesfor practical-scale
enzymaticcatalysis.
Introduction
The NAD(PXH)-requiring oxidoreductasesare porentially
useful catalystsin chiral synthesis.r.2Practical use of this class
of enzymeshas been inhibited by severalfactors: the cost of the
enzymes, the requirement for efficient cofactor regeneration
proccdures,and thc frequent requirement for opcration using dilute
solutionsof reactantsor products. [n addition, oxidoreductasecatalyzed reactionshave often proved lesseffrcient on a preparative
scale than might have bcen expccted from analytical-scalereactions. The reasonsfor this inefficiency have not been clearly
defined, and one function of this paper is to suggcstthe importance
of product inhibition in determining efficiency.
The cost of an enzyme used in synthesisis a function both of
its initial c6t (that is, the c6t to purchaseor prepare the enzyme)
and the turnover number (TN = mol of product/mol of cnzyme)
achicvedin reaction. For large-scalepreparations,reactor size
and productivity also become important. Improved methods of
enzyme stablization, especiallyimmobilization in suitablc polymer
matricesl or on solid supports,ahave dramatically increasedthe
lifetimes (i.e., turnover numbers) obtainable for the oxidoreductasesunder the conditions used for organic synthesis. Regeneration of NAD(P)H from NAD(P) is now relatively
straightforward.5'6 Thc rcverseregeneration-that of NAD(P)
from NAD(P)H-remains a more difficult problem for three
reasons: first, most enzymatic oxidations are thermodynamically
unfavorable; se@nd, oxidations are often strongly inhibited by
products; third, many of the organic oxidants of potential use in
enzyme-qrtalyzd oxidations are unstable at the higher pH values
required for maximal activity of the enzFnatic catalysts (pH =9).
t Harvard Univcnity.
( I ) For rwiew articlcs, scc: Findcis, M. A.; Whitcsidcs,G. M. Aruru. Rep.
L{ed. Chcm. l9S, 19,263-272. Whitcsidcs,G. M.: Wong, C.-H. Aldrichimica Acta 19t3, 16, 27-34. Jones,J. B. In 'Asymmctric Synthesis";
Morrison. J. D., Ed.: Acadcmic Press: Ncw York, 19t5.
(2) Hirschbcin,B. L.: Whitcsidcs,G. M.
"/. Am. Chem. Soc.19t2. 104.
445t-.1450. Wandrcy, C.: Buckmann,A. F.; Kula, M. R. Biotech. Bioeng.
t9tl. 2J. 2789-2802.
(3) Pollak,A.: Blumcnfeld,H.; Wax, M.; Baughn,R. L.; Whitcsidcs,G.
M. J. Am. Chem. Soc. 19t0. 102. 63244336.
( 4 ) K f i b a n o v ,A . M . S c i e n c e1 9 t 3 , 2 1 9 , ' 1 2 2 - 7 2 7 . C h i b a t a . I . t n
'lmmobilized
Enzymcs-Rcscarchand Dcvclopmcnt":HalstcdPress:Ncw
York. 1978.
(5) Shakcd, Z.; Whitcsidcs.G. M. J. Am. Chem. Soc. l9t{t. /02.
7 1 0 4 - 7 1 0 5 . L c v y , H . R . ; L o c w u s ,F . A . ; V e n n c s l a n dB, . I b i d . 1 9 5 7 , 7 9 .
2949-2953.
( 6 ) W o n g ,C . - H . ;W h i t e s i d c sG, . M . J . A m . C h e m .S o c .1 0 3 , 4 8 9 f 4 8 9 9 .
0 0 0 2 - ? 8 6/ 835/ I 5 0 7 - 6 9 9 9 550011 0 e
SchemeI. Methodsfor rn Situ Regeneration
of NAD from NADH
R^CHOH
R^CO
z
\
l1
t
/luuyno
a
NAD NADH
l./
,Yqntmo
\
AH
A
'.
I
,'-<,
62
Z
t'.o,
t
-vAvivxTo
F M N/ 0 2 :
[:
ir
x vo
\rAYiY
AH = \4.\."
-V]."\i,
o
(Fnr )
i Flr{x
2)
Raondofbn rrth 02
:
2-Oxoglul,rofe/G|DH
[:
-o
*9- - o-. ",;
o
o
AH=
(2-gro{lulrd}
"J!-^-".
o
o
(r-lrsrftrr)
glutomal.
d.tt@aroto
tm.2.
No raordotion
wllfi 02
.n
\OIOH|
Methylcneblue/ ctiophorosc
3
[:
Zviv\
-i/.ex.\Ar
aYN)2\
AH = -"$rAy',-
l
(|a0)
rxEH)
on.t/ma 2 s dloCtotao
Rooridotioo rith 02
The regenerationof NAD(P) from NAD(P)H is important in
both analytical assays(nmol of substrate) and in preparative
enzymatic syntheses(mol of substrate).7'8Analytical-scaleregenerationmethods are highly developedand include enzymecatalyzd regencrations,t'eoxidations by dioxygcn with electrontransfer reagents,l0stoichiometric chemical oxidations,Eand
(7) For reviewarticlcs,s€e: Lowc. C. R.; Piil. Trans. R. Sor. I-ond. Ser.
8 t9t0, 3m, n5-353. Wang S. S.; King, C.-K. In 'Advanccsin Biahenucal
Enginccring-;Springcr-Vcrlag:Bcrlin, 1979:Vol. 12, pp I l9-146.
( 8 ) P i n d c r , S . ; C l a r k , J . B . ; G r c c n b a u m ,A . L . I n " M c t h o d s o f
Enzymology';McCormick, D. 8., Wright, L. D., Eds.;AcadcmicPress:New
Y o r k . l 9 7 l ; V o l . l E B ,p p 2 f 3 0 .
(9) For the uscof malatcdchydrogenasc.
scc: Campbcll.J.; Chang,T M.
S. Eixhem. Biophys.Res.Commun.1916,69,562-569.For uscof NADH
o x i d a s c s, c e : S c h m i d t ,H . L . ; G r e n n e r ,G . E u r . J . E i x h e m . 1 9 7 6 , 6 7 .
295-302.
( 1 0 ) G r e c n b a u mA.. L . ; C l a r k ,J . B . ; M c L c a n , P .B i o c h e mJ.. 1 9 6 5 . 9 5 .
I 6 l - 1 6 6 . S m i t h . P . J . C . N a t u r e ( l t n d o n \ 1 9 5 1 .1 9 0 . 8 4 - 8 5 .
1985 American Chemical Societv
7000 J. Am. Chem. Soc., Vol. t07, No. 24. I9g5
electrochcmicaloxidations.rr To date, regenerationby dioxygen
with flavin mononucleotidc(FMN)r2 as the electron-transfer
reagent has bcen the system most often used for preparative
enzymatlcsynthesis.oxidation with 2-oxogluuratecatalyzedby
glutamatedehydrogenase
(GIDH) has beendemonstratedto be
useful,l3but has not beenextensivelydeveloped.
The goalsof this work were to ideniifv the ir{nD regeneration
systemmost suited to large-scalework, and to identify the most
i m portant factors-w hether connectedwit h cofactor regeneration
or not-that limit applicationof oxidoreductases
as catalystsin
synthesis.our approachto the first problem was to considerthe
regenerationmcthodsusedin analytical-scalework, to test likely
candidatesin preparative-scalereactions,and to modify these
methodsto meet the requircmentsfor syntheticpracticalit-v.The
criteria by which the usefulnessof the regenerationmethodswere
measured were cost, rate of regeneration, turnover number
achievedfor NAD, and simplicity of execution(in operation of
the system,in monitoring the progressof the reaction.and in the
workup). Analytical methods which we judged to be unlikely
candidatesfor immediate preparativeuse are mentioned in thl
following discussion,but were not tested experimentally;these
includcd several chemical methods and most electrociremical
oxidations.
The current preparativeregenerationmethodscan be judged
and c-omparedby thesecriteria. The method most wideiy uied
g1d1loroughly developedis thar of Jonesand Taylor.r2in which
NADH is convertedto NAD by direct reactionwith oxidized
flavin mononucleotide(FMN), and the resulting reducedflavin
(FMNH2) is reoxidizedto FMN by dioxygen(Siheme I). This
schemehas beenusedsuccessfullyin preparationsof ketoneson
a 2-g scale.ra The advantagesof the procedurebascdon FMN/O2
are that it requires no enzymesfor regenerationand that it ii
simple to carry out. Its seriousdisadvantage(aside from the
incompatibility of certain enzymeswith 02) is that the rate
constantfor reactionof FMN with NADH is low (k, = 0.2 M-r
s-r at pH 3;.ts This low rate @nstantrequircsthat conccntrations
of FMN and NAD be high to achieve useful rates. A typical
publishedreactionconrainssubstrate(2 g, l4 mmol), NAD-(0.7
g, I mmol), and FMN (9 g, 20 mmol).ra The very high ratio of
FMN to substrate complicatesproduct isolation and usesthis
speciesalmct stoichiomctrically(TN = 1.4 for FMN). The high
concentrationof NAD resultsin inefficient usc of this expensive
cofactor (TN = 24). Following the progressof the reaition is
both inconvenientand inaccurate: a portion of the reaction solution
is extracted with an organic solvent and the contents of the exrract
analyzed by GLC. [n ccrtain cases,inaccuraciesinherent in this
method lead to lowcred yields and lowcred enantiomeric excess
(vo ee).t6 The result of thesc difficulties is an uppcr limit on the
scaleof the reaction; no more than -20 mmol ol any subsrare
has bcen oxidized using this method.rT
The advantagesof thc 2-oxoglutarate/GlDH regeneration
method over FMN lo,2are scrrcral. Thc maximal rate oloxidation
(l l) Moirour. J.; Elving,P. J. Arul. Chem. yfn, SI,346-350. Aizawa,
!!.. p_oglfiq, R. !V.; Charla, M. Btuhtm. Biophys. Acta tTtS, j8S,
362-370. Moiforu. J.; Elving, p. J. J. Am. Ctum.sd. t-900. 102.653H.538.
(12) Joncs,J. B.; Taylor, K. E. Can. J. Chem. (n6, Sl,2969-29].3.
._..(.13).!Voo&C.-H.; PoilalqA.; McCurry, S. D.; Suc J. M.; Knowles,J. R.;
Whitcsidcs,G. M. Mct.h.ds_Eylmol. igt2, g9, l0t-121. Wong, C.-H.j
McCurry, S. D.: Whitcgidcs,G. M..1. lm. Chcm.Soc. lfit. I02. :'g3g-7g3g.
_-(14) Jakovac.I. J.; Goodbrand,H. B.; Loh K. p.; Joncs,J. B. J. Am.
Chcm..Soc.t9t2, 101, 4659-$55.
(15) Joncs,J. B.; Taylor, K. E. Can. J. Chcm. ln6., 51,2974-29t0.
(
. . I q) An examplein which ingccurareanalysisof the extenr of reaction.R,
lcd to lovcrod cnantiaclcctivity is thc HlADH-catalyzcd oxidation of racemic
crs-3,5dimctbyltetrahydropyran-2-ol
to cis-(3s,5R)-3,sdimcthyltctrabydro
pyran-2-one. ln order to achievemaximal % cc of both tbc unreactcd icctal
l$ =tnr Pl.odqg, exactly Sffi of thc racsrnic starting matcrial must be oxidizcd
(R 0.35). Thc low enantiomcriccxccss(35% cc) of thc unreactedstarting
matcrial was attributcd to an inaccuratcGLC analysis(which indicatcd R
= 0.5) rcsulting from 'inco-mpletccxtraction of the surting diol".
Thc product
of thc oxidation, the 3.s,5R lactonc, was cnantiomcricailypurc
1t00% e.c),
t-4ic1tiqg
oxi&tion wascomplcrelysicr'ecctcctivc. Scc:
!!c gl'y'qcaqlyz,d
Ng,-G.S. Y.; Yuan, L.-C.; Jakovac,I. J., Joncs,J. B. Tetrahedron1911,40,
t235-t243.
( I 7 ) D o d d s .D . R . ; J o n c s J, . 8 . , C h e m .C o m m u n . 1 9 t 2 .t O g f I 0 g L
Lee and Whttestdes
o f N A D H i s a c h i e v e da t l o w c o n c e n t r a t i o nos f t h e c o f a c t o r .
t u r n o v e rn u m b e r sf o r N A D o f 5 0 0 - 1 0 0 0 a r e r o u r i n e . r T h e
progressof the reactionis easilyand simply monitoredbv enzvm a t i c a n a l y s i so f 2 - o x o g l u t a r a t e . r 8T h e c o s t p e r m o l l o l i o x o g l u t a r a t ei s l e s sb y a f a c t o r o f l 5 t h a n t h a t f o r F M N ( 2 o x o g l u t a r a t e$: 1 2 / m o l ;F M N : S 2 0 0 / m o l ) . r e T h e m e t h o dr s
applicableon scales)0.1 mol.rr The disadvantages
of this method
relativeto FMN/02 8rc two: it generatesan organic product
(glutamate)and thus complicatesworkup: it requiresa second
e n z y m e( G I D H ) a n d t h u s i n c r e a s ecso s r s .
Regeneration
of NAD using2-oxoglutarate
appearedto us ro
be superiorto regenerationusing FMN/02. A clear choicebetwe€nthe two could not, however,be made until the two methods
were@mpareddirectlyin the sameprepararive,
enzyme-caralyzed
o x i d a t i o n . T h i s c o m p a r i s o n .u s i n g h o r s e l i v e r a l c o h o l d e ( H L A D H ) c a t a l y z e do x i d a t i o no f c j s - 1 . 2 - o c t o _
hydrogenase
'here.
hexanedimethanol
to the chiral t4.3.0] lactoneis described
There are, in principle.severalpotentialadvantagesto usrng
electron-transfer
reagentsto regenerateNAD. Dioxygenor an
anodewould be the ultimate oxidants(the electron-transferreagenr
and NAD would be used catalytically). In this work we have
examinedonly systemsusingdioxygenas the oxidant. The as_
semblyand workup of reactionswhich consume02 can, in prin_
ciple, be inexpensiveand uncomplicated.The most important
potentialdisadvantages
of dioxygen-based
systemsare the sensitivity of many enzymestoward dioxygen and its reduction
products,the slow ratesof many electron-transfer
reactionsrnvolvingdioxygen.and the lack of simplemethodsto monitor the
progressof certain of thesereactions. we have examined several
systemsbasedon electron-transferdyes. In our opinion.the best
of these proceduresuses 02 as the ultimate oxidizing agenr.
methyleneblue as the intermediateelectron-transfer
agent,and
diaphoraseto catalyzethe reactionbetweenmethyleneblue and
NADH (SchemeI).20 The initial problem which must be addressedin designinga useful systembasedon a catalvtic dve is
that of achievinghigh rates of reaction at low conc.nirutions of
NADH and of oxidized dye. The regenerationsystemmust be
able to oxidize dilute NADH rapidly using dilute dye for two
reasons.The fint reasonis economic: the nicotinamidecofactors
shouldbe usedin small quantititesto minimize their contribution
to the cct of the system. The secondreasonis easeof purification;
product isolation is most convenient if the concentrationsof
NAD(H) and dye are kept at low values. The requiremenrfor
dilute solutionsputs a minimum value on the rate constant for
oxidation of NADH in order that this step nor be rate determining.
For example, if an NAD turnover number of 1000 is to be
achieved,the concentrationof NAD in a solutioncontaining0.1
M substratewill bc 0.1 mM. To achievea useful rate of overall
reaction(e.9.,a rate which will oxidizea 0.I M solutionof sub.
strate in l0 h), a dye conccntrationof I mM (lVo that of the
product) requiresan effective bimolecular rate constant for the
reaction betweenoxidized dyc and NADH (whether enzymeelzlyzrd or not) of 30 M-t s-r. For comparison,the rate consrant
for the reaction of FMN with NADH in the procedureof Jones
and Taylor is tr = 0.2 M-t s-l at pH 8. This low value underlies
the high con@ntrationsof both FMN and NAD required in this
schcme.
Regeneration sequencesrelated to MB/diaphorase and
FMN/O2 have beenuscd prwiously (using phenazinemethculfate
I dichloroindophenolwith
( PMS),'0 ferricyanide and diaphorasc,2
PMS,e and methylcne blue)e'mbut only on an analytical scale.
This paper summarizeskinetic data neededto optimize methds
(18)
H. U. ln 'Methods of EnzymaticAnalysis",2nd ed.;
_Bcrgmeyer,
Verlag Chemie Weinhcim, Academic Press: New york, 1974. For specific
as:lls, sf: ! 1,577(2-oxoglutaratc),
p l50a (oxalacctatc),p 2048 (NAD),
p 2 0 5 3( N A D H ) , p 5 5 0 ( G I D H ) , p a 2 8( H L A D H ) , p a 5 8 ( C - 6 - p D H ) . T h e
assayfor G-6-PDH was uscdfor GIcDH, substitutingglucoseand NAD for
glucosc-6-phosphatc
and NADP, respectively.
(19) Priccsare from a Sigma ChcmicatCo. catalog,1984.
(20) Viche.H. G.; Fuks,R.; Rcinstcin,M. Angew.Chem. 19G4.76, Sj |.
( 2 1 ) W a l l a c c , T . C . ; C o u g h l i n , R , W . B i o t e c h .B i o e n g . l g l t , 2 0 ,
1407-1419.May, S. W.; I-andgraff,L. M. Biuhem. Biophys.Res.Commun.
1 9 1 6 .6 8 . 7 8 6 - 7 9 8 .
In Situ RegenerationoJ'.\AD from iVADH
I
J . A m . C h e m . S o c . ,V o l . t 0 7 , . \ o . 2 1 , t q g 5
basedon electron-transfer
dyesand demonstratesMB/diaphorase
regeneratiow
n i t h t h e H L A D H - c a t a l y z e do x i d a t i o no f c i s - 1 , 2 cyclohexa
nedimethanol.
our decisionto demonstrateNAD regenerationwith the oxidation of cis-1,2-cyclohexanedimethanoi
to the bicycliclactone
was basedon the effectiveirreversibility
1V,1V,= 150)and favorablekinetic parameters(K,"/K, = 0 3)2?of the reaction(Vr
and v, are the maximal ratesof the forward and reversereactions
per mg of enzyme:K. is the Michaelisconstantfor the substratc:
K , i s t h e i n h i b i t o r c o n s t a n to f t h e f i n a l p r o d u c r .( + ) - ( l R . 6 . 5 ) _
cis-8-oxabicvclononan-7-one).2r
Argumentsdetailedlater in this
paper establishthat oxidationsusing most dehydrogenases
for
which K^l K, ) I give low yields of product ,egardlessof the
method of NAD regeneration,becausemost of thesereactions
are characterizedby noncompetitiveproduct inhibition (or kinetically similar mixed inhibition). Thesetypesof inhibition result
in a decreasein the rate of the forward reactionas product is
formed. This inhibition cannorbe overcomethroughihe useof
a more powerful oxidant or by high conc€ntrationsof subsrrate;
although the overall reactionwill be thermodynamicallyfavorable.
the low reactionrateswill result in unacceptablylong reaction
times. The only effective methodsfor minimizing thJeffects of
noncompetitiveor mixed product inhibition is to remove the
product from the reaction systemas it is formed (for example,
by extractioninto a secondphase).2a
The HlADH-catalyzed oxidationof crs-cycrohexanedimethanol
to the bicyclic lactone is also attractive as a demonstrationsysrem
becausethe method providcsa chiral product which is not easily
obtained by nonenzymaticmethods. There are now other, simpler
methodsto obtain the chiral lactone(using,e.g.,pig liver esterase).25 We wished,however,to comparettir typi Jf procedure
with a numbcr of other enzymaticmethodsfor generaiingchiral
centers.and the large body of useful information developcdby
Joneson chiral oxidationswith HLADH providesan important
comparison.
Results and Discussion
NAD Regenerationwith 2-Oxogluterete/GlDH. [n our expcrience, this method is the most useful now available for regeneration of NAD from NADH. It requires ammonia, 2-oxo_
glutaratg and glutamatedehydrogenase
(GIDH). The Expcrimental section details a procedure for the oxidation ol ciscyclohexanedimethanol( I ) to (+)-( I R,6s)-cis-g-oxabicvclononan-7-one(2) on a 70-mmol (10-g) scaleusing 2-oxoglutarate
()^o'
\,A',oH
r.
lo
frntaox
NAD+
NADH
p,un-oh
NHI
- A
|
{
doffioganaso
(t)
\O
"
.-oftooo06
.-oft'o-.
z NH;
(22 il and NAD (0.1 g) in a mixture of water (0.g L) and hcxane
( I L). Thc reaction was carried out using solublc HLADH and
immobilized GIDH in a l:l v:v mixturJ of watcr (pH g) and
hexane. The progressof thc reaction was followed by periodic,
enzymatic assayof unrcacted 2-oxoglutarate. The reaction *as
complete in 3 days and the lactone 2 was isolatedingsvo vield.
(22) Lcc, L. G.; Whitesidcs,G. M., to bc submittcd for publication.
(23) In this manurrip, K; refcn only to thc conccntrationof nonofactor
product yh-i9h,p1od99!"a velocity of v*/z
at saturating subrtratc conccn(> l0 fu. K. is not cquivalcntto tic paramctcrsio, Kl K1 (subrtratc
!.?ttgr
inhibition),etc.
(24) Martinck, K.; Scmenw. A. N.
giuhen. l9tt. J,g3_t26.
"/. Appl.
Martinck K.: Lcvashov,
{._y, K}rmclnitsky,Y. t.; Klyachko.N.'t-.: ncrcan.
l. Y. Science19t2, 2lE, t8F89l. Martinclq K.; Scmcnov.A. N.; B"..r,n,
l. Y. Biuhim. Biophys. Acta t9tl, 6jB,76-g9.
(25) Sabbioni,G.; Shca, M. L.; Joncs,J. B. Chem. Commun. l9&t,
236-238.
7001
l.O
O.5
O.4
[oa] o .3
[oa].
O.2
200
400
600
800
tooo
t200
t i m e ( m r )n
Figurel. First-order
plotof thedecomposition
(oA) in
of oxalacerare
differentbuffersolutions.Valuesof t, were: 4.2 x l0{ s-r for 0.I M
Hepeswith 0.1 M glycinc,pH 7.6(O); 1.6x l0-5s-r for 0.1 M Heoes
w i t h5 m g / m l o f s o l u b lH
e L A D H , p H 7 . 6( r ) ; g . gx l 0 - { s - ri o r b .t
M Hepes,pH 7.6(O).
The TN for NAD was 500. There s€emto be no intrinsic problems
in scalingthe reactionto larger quantities. In this procedurethe
overall rate-limiting step was the HLADH-catalyzed oxidation.
and the most expcnsivecomponentof the systemwas HLADH.
The urscof hexaneas an immiscible secondphasewas important
in that it removedthe product as it formed and thus minimized
product inhibition. The volume of the aqueousphasecalculated
to be optimal for theseenzymesand quantities of reagentswas
9.6 L." In practice,we useda slightly larger volumelwith less
favorableratesof reaction) in order to solubilizc 2-oxoglutarate
and the cis diol.27 The cis diol substratewas not sigriificantly
soluble in hexaneand the lactone product had a pariition coefficient (p) of 1.0 bctwecnhexaneand water containingglycine
buffer. The 2-oxoglutaratc/GIDH systemhas certain inlrinsic
disadvantage-especially the requirement for stoichiometric
quantities of 2-oxoglutarateand the fact that glutamate is produced. It is, however,clearly more practical than the procedures
basedon electron-transferdyes which follow.
NAD Regwatim
Usfog lrctrte netyarugemse yecst Alcohol
Dehydrogmrse' rnd Mdrte Dehydrogenese. The successof the
2-oxoglutarate/GlDH rcgenerationsystem led us to consider
related systenuibasedon other readily available dchydrogenases.
The requirements for a successfulenzymatic method are i,,"o, un
inexpcnsivedehydrogenaseand a substrate which is a stable.
inexpensiveoxidizing agcnt. l-Lactate dehydrogenaseand yeast
alcohol dehydrogenaseare both inexpcnsive, but pyruvate and
acctaldehydeare both unstablein solution, particularly at high
values of pH. In addition, pyruvic acid and its condensatron
product are significantly solublc in organic solvents and would
bc expcctcd to complicate workup. Malate dehydrogenaseis very
inerpcnsive; however, oxalacetate decarboxylateJin solution,
particularly when amincs or solubleHLADH arc pres€nt(Figure
l)' The half-life of oxalacetate(l-9 h) is unacceptabryshort, given
its high initial cost (9160/mol).re It may be possibieto develop
useful regencration mcthods basedon thesc systemsby circumventing the indicated (and other) problcms, but we have not
workcd on them as part of this research.
Oxidrtiotr of NADH by Ehcton-Tnnsfer
Dyes: Uncetelyzed
Reectioc. Rate constants for the dircct (noncnzyme catalyled;
reaction of NADH with the oxidized form of scveral dyes were
measured by following the disappcaranc€of NADH
nrn
"i3+0
(26) An cquation (rcf 22) which givcs thc optimal valuc of
[Se]/K, to
minimizc thc timc to complctc a rcaciion to cxtcnt of rcaction R foij given
valucs of K. and K1 was uscd:
? =[*'f '2h(r'*,]'
Valucsof K, (26 mM). Kr (t0 mM), R (0.95),and
[So](70 mmol) wereuscd
to calculatcthe optimal volumc(0.6 L).
(27) A parallcl rcaction without hcxanc was not pcrformcd.
1002 J. Am. Chem.Soc.,Vol. 107,No. 24, 1985
Lee and Whitesides
and Diaphorasc-Catalyzed
TebleI. Uncatalyzed
Oxidationof NADH to NAD by OxidizedDyes
uncatalyzed
poa
dyc
m e t h y l e n eb l u e ( M B )
0.01
4.44
4,22
+0.22
+0.06
+0.36
pH
k2,M-r 5-t
9.0
5 . 3r 1 0 . 5
7.6
3 . 2X 0 . 2
7.6 0
7.6 0.3
7.6
1.0
'70
7.6
7.6
2
catalyzed
u . bM s - l
x 107
sp act.
(U/mg)'
ut M s-l
x 106
l0
0.02
0.06
20
0.03
0.1
20
tA
It
8.3
0
0.8
rate of reoxidation
of reduccd dye, k,
( s - r )x l 0 r
stabilityof
dye system
m o d e r a t e ,2 . 4 + 0 . 1
m o d e r a t e2, . 4 * , 0 . 1
fast
fast
no reaction
moderate
no reactton
stable
m e t h y l v i o l o g e n( M V )
stable
f l a v i n m o n o n u c l e o t i d e( F M N )
unstabled
d i c h l o r o i n d o p h e n o(lD C I P )
J
r0
stable
p h e n a z i n em e t h o s u l f a t e( P M S )
200
e
unstable
p o t a s s i u mf e r r i c y a n i d e( K 3 F e ( C N ) 5 )
5
r0
20
stable
o R e f e r e n c e3 4 . 0 T h e c a l c u l a t e dr a r e p r o d u c t i o n
of
o f N A D f r o m NADH for theseconcenrrations:o x i d i z e dd y e , I m M : N A D H . 2 . 6 x l 0 { M :
d i a p h o r a s e( i f p r e s e n t )1 0 0 m g / L . . C o n c e n t r a t i o n sa s s u m e dw e r e : d y e . 0 . 2m M ; N A D H , 0 . 2 4 m M . U n c e r t a i n t i e sa r e * 2 0 V o . d I n " H a n d b o o k o f
B i o c h e m i s t r y " ;S o b e r . H . A . ; E d . ; C R C P r e s s : C l e v e l a n d ,1 9 7 0 rp p K - 4 1 - K 4 2 . ' N o t m e a s u r e d .
O.2O
Oel6
a
traed
8
va
y
"\
[Me]
Ool2
(mM min-t )
o4E
O17
0.6
o
30
60
r5o
t?.'n,
r8o 2ro
l::
Figure2. Pseudo-first-order
plot of thc oxidationof rcduccdmethylene
blue(MBH) by oxygenat pH 7.6(O) andpH 9.0(O). For borh,k, =
( 2 . 4+ , 0 . 1 )x l 0 - rs - r .
spectrophotometrically.Results are summarized in Table I.
Oxidationsby methyleneblue were followedcarefully at two values
of pH. and at scvcral concentrationsof methylene blue and NADH
between0.06 and 0.25 mM. Oxidations by other dyes were
followedonly at a dye concentrationof 0.2 mM and an NADH
concentrationof 0.25 mM. Thesedata indicarethar FMN, the
oxidizing dye employedin the presentlyuscd proccdure for direct
reoxidation of NADH to NAD. is significantly slower than several
of the other dyes examined (methyl viologen,dichloroindophenol,
phenazinemethosulfate).2e These rates are not. of course,thc
only parametersof interestin choosinga reoxidationsystem. In
particular. the rate of reaction of the reduceddye with 02 must
also be rapid to achievehigh ovcrall rates, and the dye must be
stable under the reaction conditions. Dichloroindophenoloxidizes
NADH reasonablyrapidly, but the reduccd form of DCIP is not
autooxidizable.ro PMS reacts rapidly with NADH without
diaphoraseand is autooxidizable;it has bcen used to regenerare
NAD in severalanalytical systcms. PMS is, however,unstable
under reaction conditions; it undcrgoesreactions with oxygen
which are both photocatalyzcd(yielding pyocyanine)and dark
(yielding 2-keto-N-methylphenazincand phcnazine).3r DCIP
and PMS were discarded for practical synthetic applications on
the basis of these characteristics.
Reduced methylene bluc reacts rapidly with dioxygen in a
stirred solution in contact with air. Rates of this autooxidation
were measuredat pH 7.6. Figure 2 shows the resulting pseudo-first-order kinctic plots. Rate constants are given in Tablc I.
Thesedata in combination suggestthat subotitutionof methylene
blue for FMN in the Jonesand Taylor procedurewould increase
rates and decreasethe quantity of dye required. The advantage
(2E) Pedcrson.K. J. J. Am. Chem.Soc. 193t. 60. 595-601.
(29) Thc reactionis acceleratedby light. Scc: Chambcrs.R. P.: Ford. J.
R.;Allender.J. H.; Bancs. W. H.; Cohcn.W. In'Enzymc Enginecnng':rye.
E . K . . W i n g a r d .L . 8 . , E d s . :W i l c y : N c w Y o r k . 1 9 7 4 :V o l . 2 . p p 1 9 5 - 2 0 2 .
( 3 0 ) G u r r ,E . :, { n a n d N
, . : U n n i .M . K . ; A y y a n g a rN. . R . I n ' T h c C h c m istry of SyntheticDyes': Venkataraman.K., Ed.; AcadcmicPress:New York.
1 9 7 . 1V: o l 7 , p p 3 0 5 - 3 0 8 .
( 3 1 )M c l l w a i nH
. . - / .C h e m S
. o c .1 9 3 7 .1 7 0 4 - l 7 l l .
O.2
O.3
or4
v/[S]
O.5
016
Oo7
O€
(min-r)
Figure3. Eadi*Hofsteeplotsusedin rhedeterminationof
valuesof K.
of mcthylene
blucwith NADH anddiaphorasc
ar pH 7 6 (O) and pH
9 . 0 ( O ) . T h ev a l u eo f K . a t p H 7 6 w a s0 . 2 4t 0 . 0 2m M ; a t p H 9 0 ,
1.7 + 0.4 mM. The circledpointswereomittedirom the analvsis.
of this substitutionwould, however,be marginal,and balanced
by the slower rate of reoxidationof reducedmethyleneblue as
compared to the reoxidation of reduced FMN.
We concludethat none of the systemssummarizedin Table
I providea genuinelypractical procedurefor the regenerationof
NAD, becauseall are too slow. In searchingfor someprocedure
which would significantly increasethe rate of reoxidation of
NADH at low concentrationsof both NADH and dye, we explored enzymaticcatalysisof this step.
Oxidetion of NADH by Methylene Blue/O2 Catalyzed by
Diephorese. Diaphorasecatalyzesrhe oxidation of NADH by'
oxrdizcdmethyleneblue. Diaphoraseactivity is found in lipoamide
dehydrogenases
obtainedfrom many sources(e.g.,porcineheart.
yeast, and Clostridium kluyuerr). The Clostridium enzyme used
in thesestudieswas commerciallyavailableand relativelyrnexpensive. Kinetic parametersfor the diaphorase-catalyzed
reacrion
of NADH by MB were determinedusing standardprocedures.
Figure 3 shows Eadi*Hofstee plots used to determined K* for
methyleneblue. Valuesobtainedwere: pH 7.6, K*(MB) = 0.24
* 0.02 mM; pH 9.0,K',(MB) = t.7 * 0.4 mM. It was more
difficult to obtain an accurate value of K. for NADH. since it
was low, Examinationof ratesof oxidationof NADH at pH 7.6
over the concentrationrange 2 x l0-5 to 1.8 x l0-l M with a
methylencblue concentrationof 0.6 mM (approximatelytwice
,(,'.,(MB)) shows no variation in rate. We thus infer that K,"(NADH) is lessthan lf5 M. An NAD regenerationsystemwas
demonstrated with the cnantioselectiveoxidation of cis- 1,2cyclohexanedimethanolcatalyzed by immobilized horse liver
(HLADH) at pH 7.6. The best results
alcoholdehydrogenase
were obtainedin a two-phasesystem;hexanewas again usedto
removethe lactoneselectivelyfrom the aqueousphase.l2 Oxygen
was slowly bubbled through the mixture. The reactor color
( 3 2 ) H L A D H h a sa l s ob c c nu s e di n h o m o g e n e o us so l u t i o nos f w a t e ra n d
organicsolvents.Scc: Jones,J. B.: Schwartz,H. M. Can.J. Chem. 1982.
60.1030-1031.
In Situ Regenerationof NAD from NADH
J. Am. Chem. Soc Vol. 107, ,\o. 24, 1985
changedfrom an initial deep blue to colorless:then, after 24 h,
back to deep blue. The diol (4 g, 28 mmol) was convertedto
lactone(3.2 g, 83Vo,l00Voee) in 4 days. The TNs for NAD and
for methyleneblue were 350 and 580, respcctivcly.
This procedureprovidesa practical method for regenerating
NAD from NADH in situ. It is marginallysuperiorin convenience and practicality to the procedurebasedon FMN/O2.'2
Much smaller quantititesof dye are used.and the reactionvolume
is smaller. but the systemis renderedmore complex and expensive
by the requirementfor an additionalenzyme(diaphorase).For
FMN/O:. the sloweststepis reactionof FMN with NADH: for
MB/diaphorase/O2,the sloweststepis reactionof 02 with MBH.
Although the latter reacrionis slightly faster than the former,
FMN is much more solublein water than MBH and the effective
rates are higher.
For comparison with the 2-oxoglutarate/GlDH method.
preparationof l0 g of lactoneby oxidationof cis-cyclohexanedimethanolusing MBlO2ldiaphorase would require NAD (0.25
g) and methyleneblue (0.08 g) in a mixture of water (0.25 L)
and hexane( 1.3 L) and would require a reactiontime of 4 days.
A synthesisof 2 on the same scale using FMN/O2 would be
extrapolated from literature data to require NAD (3.5 g) and
FMN (45 g) rn 5 L of water,l3and a reactiontime of 3 days. Thus
the methodsbasedon 2-oxoglutarate/GlDHand MBlOrldiaphorase require smaller volumes. The use of smaller volumes
simplified isolationof the product and, in the caseof the 2-oxoglutarate/GlDH method, resultedin a workable isolationof residual HLADH by ultrafiltration. The method basedon 2-oxoglutarate/G|DH is intrinsicallycleanerthan the FMN/O2 system.
The product in the first caseis isolatedas pale yellow oil; with
FMN/O2 regenerationit is isolatedas 'black oil" presumablydue
to decompositionof FMN. The 2-oxoglutarate/GlDH method
allows the reaction to be followed simply and accurately by assaying residual 2-oxoglutarateenzymatically;this caseof analysis
is a significant advantageover the FMN/O2 method when
achieving a high Vce,, for the product depcndson accurate analysis
of R.r6
The method based on MB/O2/diaphorase does not provide
significantlyfaster rates than the FMN/O2 system. Smaller
amounts of MB than FMN are required to achievesimilar rares;
however,MB is slightly soluble in organic solventsand workup
of reactionsusing MB/O2/diaphorasc requiresan additional step
(filtration through charcoal).
NAD Rqenendoo with Ferricyuride Crtrlyzed by Dilphorese.
The slow rate of oxidation of reduced methylene blue by 02 in
the dye-basedsysternsled us to examine Fe(CN)eF as the terminal
oxidant. This speciesis inexpcnsiveand very water soluble,and
reacts rapidly with NADH (Table I). The reduced form, ferrocyanide(Fe(CN)6F), docs not react with dioxygen,and 2 equiv
of the one-electrontransfer reagent is uscd for each two-electron
oxidation. The reactionof Fe(CN)rF with NADH is also catalyzed by diaphorase.2r
This NAD regeneration system was not tested using the oxidation of the cis diol t, but rather with the conversionof glucose
to gluconate (eq 2) becausethe oxidation is not complicated by
7003
o
5
Bose
0dde0
( m L)
A
4
Ao
3
(Extent
of
Reocfron)-l
?
QtZ
O.O
ot23456
T r m e ( h)
Figure 4. Two methodsusedto follow the progressof glucoseoxidation.
P l o t o f K O H ( 2 . 5 N ) a d d e dt o m a i n t a i nn e u t r a l i t yv s . t i m e ( o ) a n d p l o t
o f t h e r a t i o o f a b s o r b a n c ct o i n i t i a l a b s o r b a n c ea t 4 2 0 n m v s . r i m e ( t ) .
T h e r e a c t i o nc o n t a i n e dg l u c o s c ,N A D , K r F e ( C N ) c , G l c D H , a n d d i a phorase.
l.o
O.8
A
a
Or6
AA
v
vo
or4
.A
n
n
Oo2
,A
OoO
0.0
0r4
O.5
| e.
te6
lonrc Strengfh (M)
Z&
Figure 5. Effect of ionic strength on the activities of HLADH in the
oxidation of cis-cyclohcxancdimethanol
and GIDH in the reducriveamination of 2-oxoglutaratc. The symbols O and a represenractivity of
GIDH with added NaCl and Na2SOa,respectively.The symbolsO and
A respresentactivity of HLADH with added NaCl and Na2SOa,resp€ctively.
product inhibition by gluconateand the extent of reaction R is
easily monitored by measuring the volume of added base required
to maintain the pH at 7.0. In addition, glucosedchydrogenase
(GlcDH) is easily immobilized (comparedto HLADH). We
hoped that this characteristicmight help to retard denaturation
of the enzyme by Fe(CN)rF. This oxidation was conductedonly
on small scale (5 mmol of glucose),using NAD (0.03 g) and
immobilizeddiaphoraseand GIcDH in 0.07 L of water. Thc
reaction was conducted on a small scale bccauseof the large
reaction volumesrequired in order to keep the ionic strength of
the solutionsat reasonablylow values (see below). The progress
of the reaction was followed by two methods: moniroring the
quantity of baseadded to maintain the pH at 7 and measuring
the decreasein absorbanccat 420 nm, c.rr",of Fe(CN)rF. Figure
4 showsplots of the resultsof both methods. The reaction was
completein 6 h; the TN for NAD was 125.
The uscfulnessof stoichiometric ferricyanide NADH regeneration for largc-scalesynthcsisis limited by the high ionic strengrh
of a solution of thc rcagcnt. Many cnzymes have dccreased
activitics at increascdionic strength. Figure 5 plots the activity
of HLADH and GIDH at various valuesof ionic strengths using
two electrolytes: NaCl and Na2SOa. The ionic strengrh of a
solution of potassium ferricyanide is eight times its molarity; of
potassiumferrocyanide,l0 times its molarity. The rate of oxidation of crs-cyclohcxanedimethanol
at an initial substrateconcentration of 70 mM (5K.) would be only 20Voof the optimal
rate. In addition, ferricyanideis a potent oxidizing agent (E." =
+0.36 V)ra and high concentrationsmay oxidize thiol moieties
and deactivateenzvmes.
(33) Thac numbcrsarc bascdon literatureproccdures:it is not clear why
such dilute solutionsare uscd or if thcy are actuallv nccdcd.
(3a) Lmch. P. A. In 'Handbook of Biochemistry";Sober,H. A.. Ed.;CRC
Press: Cleveland.1970;pp J-33-J-a0.
xoSS|3te,
Ho\/
xor-(Pox
J\foT
o'..
Xgu*ro
rrl
ilAD?
NADH
k
.-
2 Fc(CNf-
dohteoeonoto
(2)
dostq?se
-z
Fc(cN).s-
fl
7004 J. Am. Chem. Soc., Vol. t07, No. 24, t9A5
Lee and Whitestdes
f)emonstrationof Product Inhibition. we stated earlier that
the HLADH-catalyzed oxidationof cis-cycrohexancdimethanol
to the chiral lactoneis a useful reactionwith which to demonstrate
NAD regenerationnot only becausethe reaction is irreversible
but becausethe product is a poor inhibitor. If thermodynamic
irreversibilitywere the only criterion for successfulreaction,addition of acetaldehyde-the productof HLADH-catalyzed oxidation of ethanol-to a reactionmixture containingciidiol and
HLADH should not affect the rate of productionoi the lactone.
In fact. acetaldehydeinhibits oxidationof the cis diol to the same
extent that it inhibits oxidationof ethanol;the valuesfor K; for
acetaldehydewere the same for both oxidations(K, = I mM at
pH 9)
Influence of Product Inhibition on Syntheses cstrlyzed by
oxidoreduct$€s. Even with a good cofactor regenerationsysrem,
*1ny (perhap mmt) oxidationsof simple alcoholsto ketones(and
related transformationsusing dehydrogenases)
will remain problematic. The fundamental difficulty in this area often lies not
in cofactor regenerationbut in inhibition of the participating
oxidoreductasesby the products of the reactions. ntt-troug[
product inhibition can sometimesbe circumventedby removing
the product as it forms (either by physicalmethodssuch as extraction or by further chemicaltransformationin casesof noncompetitiveor mixed inhibition,or by usinghigh concentrations
of substratesin casesof competitiveinhibition), such methodsare
not always convenient.
Many oxidoreductaseswhich catalyze the oxidation of alcohols
(yeastalcoholdehydrogenase,
horseliver alcoholdehydrogenase,
glyceroldehydrogenase.
lactatedehydrogenase)15
are belilved to
follow ordered bibi mechanisms.16The systemshows mixed
N+0
R2CftOrl
RzC-O
iill
,t12L
Enz Enz-NAD dr^n*o^
\ruao
+E;
NADH
_l)
\Haox
Enr- NADH Enz
in_hibition
by product ketone. This inhibition reflectsa binding
of ketone to th€ Enz-NADH complexeswhich has the effect ol
lowering the apparent@ncentrationof enzyme. This inhibition
cannot, as with competitive inhibition, b€ overcomesimply by
increasingthe concentrationof alcohol.
The use of theseoxidoreductasesfor large-scalesynthesiswill
be practical, regardlessof thc availability of an effective NAD
regenerationsystem,only if the kinetics of the fundamental rcaction (eq l) are favorable under conditions which might be uscd
NAD +R2CHOH:
R 2 C = = O+ N A D H + H +
(3)
K ' , n= l 0 7 K , q= [ R , C H O H ] t N A D l / [ R 2 C : O J I N A D H ]
(4)
in a plausiblesynthetic procedure(that is, relatively concentrated
solutionsof reactants and products). The most important parametersin determining the practicality of an enzyme-catalyzed
oxidation are the Michaelis constantfor the alcohol (K.nzcuoH;
and the inhibition constant for the ketone (K,Rrco). Thc concentration of NAD can bc maintainedgreater than K'NAD lz gt
choosingappropriate starting conccntrationsof this cofactor and
maintaining efficient cofactor recycling. The constant (,R:co
indicatesthe quantity of ketonewhich lowersthe maximum velocity of the oxidation by half at kinetically saturating conccntrationsof NAD and R2CHOH. Its magnitudedeterminesthe
maximum concentrationof product which can be achievedin the
reaction before the rate of reaction drops to an unacccptably low
value due to product inhibition. The constant KrRlcHoH is the
quantity of alcohol which produceshalf the maximal velocity at
saturating concentrationsof NAD in the abe€nceof any products.
Its magnitudedeterminesthe minimum conccntrationof alcohol
required to achievea forward rate which makes acceptableuse
(15) Dalziel.K. In 'The Enzymes",3rd cd: Boyer,p. D.. Ed.: Acadcmic
P r e s s :N c w Y o r k , 1 9 7 5 ;V o l . X I , p p l - 6 0 .
( 1 6 ) S e g e l .L H . l n ' E n z y m e K i n e t i c s "W
; i l c y : N c w y o r k . 1 9 7 5 ;p p
5 6r - 5 9 0 .
137) Valucsof K, for NAD for the cnzymcsin Tablc II rangefrom l0-7
to l0-" M.
-3
-2
-t
|
0
2
log (Kr,mRrco
/ Km/Rrcxox)
3
Figure6. Plot of log ,<'* vs. log 1K,..R:co7K..,R.toH)
for NAD(p)_
(H)-dependent
dehydrogenas€s.
Thesymbolo represcnts
oxidations
with
NAD(P)+;thesymbolO represents
reductions
with NAD(p)H. ,K* is
definedin eq 4l thesamenumberis usedior bothoxidativeandreductive
reactionsof eachenzyme-catalyzed
transformation.For oxidations.
,(,.,n|r^t_o/K.nMo*refers ro K,RrcoTKmRcHoH.
for reducrions.
K,,*tto/K,n.RcboH
refersto KrRzcoTK,RcHoH.
Dataare from TableII
andarc fittcd to thecquationK'^ = c(K,^l K^,). Pointsare rabered
with
numbcrscorresponding
to reaciionsin Table II. The valueof c for
oxidations
(1.3
+ 0.7)x l0-r. Thevalucof c for reductions
is
is (7.9t
l) x t0-r. The circledpoint,whichrepresenrs
malatedehydrogenase
catalvzedreductionof oxalacetate.
wasomrttedfrom the analysis.
9f lh:..9q!"lytic potential of the enzyme. If the ratio K,R'co/
(rR:cHoH ( l, the reaction."n nru.i proceedefficiently-that
is, with both high catalytic ratesand high conversionsof reacranrs
to products-because concentrationsof reactant high enoughto
saturatethe enzymeactive site will produceconcentrationsof
product_highgn_gu_gh
to be strongly inhibitory at low conversions.
If KiR,co/KrR2cHoH
) l, the reiction can. in principle.proceed
to acceptableconversions.Even if KiRfo/K-RfHoH ) l, however.
the reaction may still not be practical i; g nrco is low, because
only dilute solutionsof product can bc generatedwithout serious
inhibition of the enzyme.
Sincc knowledgeof both the ratio K,R,coTKrR?cHoH
and the
individual constants K'*, K,Rtco, and (.RrcHox are useful in
evaluating an enzyme-catalyzedoxidation (or reduction), we
wished to know whether it might be possibleto estimate these
parameterswithout conductingexplicit experimentalevaluations
of them. In consideringthis problem, we have discoveredan
interesting, potentially^useful.1nd presently largely empirical
correlationbetweenK,*p/Kr*pHoH and K'* (eq 3) for a number
of oxidoreductase-catalyzed
reactionswhich'interconvertketone
and alcohol moieties(Figure 6, Table II). Figure 6 indicatesthat
within the group of reactionsexamined,K'". is linearly related
to the ratio of the Michaelis constant for the reactant and the
inhibitor constantof the produa (eq 5). The ratio of the kineric
K* = c(K,.rR,co/K..iRrCHoH)
(5)
param^e_ters
for^either oxidations or reductions is expressedas
K'.**1?-K',iRtcHoH: for oxidations it is defined as K,R:co7
K.R:cHoH,and for rcductionsit is defined as K.R,co75 n:cxoti.
Equations6 and 7 provide the empirical valuesof c for oxidations
and reductions.
K'* = [(1.3 + 0.7) x 1g-:]15rrco7g,RzcHoH;(oxidations)
(6)
K'*=
[(7.9 + 6) X l0-3]16.n,co75R:cHoH; (reductions)
(7)
The empiricalohcrvation that eq 3 conelatesdata for a number
of apparentlyonly looselyrelatedenzymaticrectionsdoesnot.
so far as we presentlyunderstand,derive directly from elemenury
thermodynamicand kinetic constants.
In Situ Regenerationof l'{AD from NADH
J. Am. Chem. Soc., Vol. t07, |'to. 24, t985 7005
Trble II Kinetrcs
andThermodvnamic
Data for Dehydrogenases
reacuon
reactlon
tb
2c
J'
1a
v
6t
7r
E'
v
R2CHOH
enzvme
malate
L-malate
dehydrogenase
g lv c e r o l
glycerol
d e h rd r o g e n a s e
l a c t at e
lactate
dehvdrogenase
y e a s ta l c o h o l
ethanol
dehy-drogenase
HLADH
ethanol
HL,A,DH
cyclohexanol
malic enzyme
L-malate
3- h yd r o x y butyrate
lsocltrate
3-hydroxybutyrate
isocitrate
(,r'rR:CHoH
R2CO
(M)
KiRrco (M)
K,R2co (M)
6'R2cHOH
(M)
K'no
oxalacetate
3 . 9x l 0 {
5 . 5x l 0 - {
8 . 0x l 0 - 5
dihydroxyacetone
9 x tO-l
3 x l0-'{
l.l x l0{
2.4 x l0-5
pyruvate
6.7x l0-r
2.8 x l0-.
1 . 6x l 0 {
1 . 3x l 0 - r 2 . 8 x l 0 - 5
acetaldehyde
l . l x l 0 - 2 6 . 7x l 0 {
7 . 8x l 0 {
4 . 3x l 0 - 2 8 x l 0 - 5
acetaldehyde
cyclohexanone
p y r u v a t e+ C O 2
5 . 5 x I 0 - ' 4 8 . 7x l 0 - 5
1 . 6x l 0 4 x l0-2
2 . 9 x l 0 - ' 4 5 . 2x l 0 - t ( p y r )
2.4 x
7.6 x
l.l x
2 . 6x l 0 - r ( c o z ) 4.5 x
4.1 x 10-<
2 . 8x
aceroacerae
l0-{
l0-l
l 0 - 2( p y r )
t0-"{(coz)
l0{
3 . 6x l 0 {
1 . 5x l 0 - 5
1 . 9x l 0 - 2 9 2 x 1 0 - 5
1 . 6x l 0 {
5.5x l0-2
3 . 1x l 0 - :
1 . 4x l 0 - l
1 . 6x l 0 - 5( 2 - o x ) 1 . 3x l 0 - { t . J
1 . 9x t 0 - r ( c o ) 2 )
a(i{ is dcfincdin rhc tcxt (eq 4). 'Kin.tic data: Hcydc,E.: Ainsworrh,S.
!. &iol. Ch.m.19{'i.213,2413-2423. Equilibriumdata: Srcrn.J. R.r
_
ochoa, S: Lyncn. F. Ibid. 1952,19t, ll3-121. 'Kinctic and cquilibriumdatd: Mccrcgor, W. G.; Phillipc.J.; Suilter, C. H. fbid. 19i4.24g,
2-oxoglutarate* CO,
1 . 2x 1 0 - 6
!r32-isg:rerzi.,xin"rica"ri,
siiiii"gr'.n.,i*i.'o.i.;;l';i;;';A:r;;.i;;::ii;;.i;"liifiil;;;;ru;fi;,.ri.'i.?ffi.;:11.
Schwcr!.C. w. /riy'. 1935,221. l9l-209. 'Kinctic d6!a: sccfmtnotcf Equilibriumdet': B.cklin. K. I. ,{.rd Crr.m. Scond.lg3t, t 2. lzig-t2ls
/Kinctic Data: Wrancn.C. C.;Cl.l.nd,W.W. Biochemiry 1963,2,915-941.Equilibrium
data: s.. footnotcg.rKincticandcquilibrium
dara:
Mctrilt. A D.i Tomkins.G. M. J. giol. Chen. 1959.231. 2118-27E2;ErpcrimcntalScction.rhis pepcr. aKinctic-data:Schimcrlik,-M.t.: Clcland.
W. w. Siochcmittrvl9n, 16,565-510. Equilibriumdata: Schimerlik,M. I.; Rifc. J. E.; Clcland,*. Vl. Ibid.ln'. 21.534?-5j54. Thc vatucof
K,/I(;rrascstimatcdbydividinsthcsquarcrmtofthcproductofth.valucsofK.ofthctwosubctmlcsbythcvelucof(iofrhcproduct.,Kinctic
daB: Bcr8mcycr.H. U.; Cawchn.K.i Klotzsch,H.; Krcb6,H. A.; Williamson,D. H. &ioch.m.J. 1967,IO2, 423-431. Equilibriumdata: Krcb6.
H. A ; Mcllanby,J.r Wifliamson,D. H. Ibid. 1962,E2, 96-98. r Kinctic data: Uhr. M. L.r Thomp6on,v. w.; Clcland.w * . J - Biol. chem. 1914.
219,2920-2921 Equilibriumda!a: Ochoa.S. In "Mcthodsof Enzymology";Colowick,S. P., Kaplan,N. C.. Eds.iAcadcmicPrcss: Ncw york,
1955:Vol. I, p 699-704.The valueof the ratioK^lK, wascalculated
as in footnote
l.
ln searchingfor a theoreticalrationalizationfor this empirical
correlationwe haveconsidereda Haldane equationfor the ondered
bi-bi mechanism(eq 8: Zr".l refers to the maximum velocity in
( r'r"rf)
a\
( K, NADH ) ( Km kctonc)
-
w
\t
(r.".r)(K,NAD)1K..t*tot;
I
o
(8)
the direction of oxidation of alcohol; Znoo.refen to the maximum
velocity in the direction of reductionof ketone;K'* is definedby
eq 4).r5 [n the courseof manipulating this equation and exploring
possiblerelationsbetweenit and eq 5, we note a secondcurious
correlation: a plot of log K'* vs. log (K,nkoonc/Kmalcobol)
for the
samc s€t of oxidoreductasc-caialyzd raclions included in Figure
6 also yields a reasonablystraight linc (Figure 7). (A plot of
vs. either log K'rk"tonoor log Kmilcobol
log K
is notably more
"q correlation
scattered:
coefficientsr = -O.8). The least-squares
slopeof Figure 7 is 1.5.rather than the value of - 1.0 observed
in Figure 6. The valuesof Km for the alcohol and ketone substrates
were. however,generally measuredat different valuesof pH; a
closer correlation may exist for a set of valuesof K. measured
at a common value of pH.
Thesecorrelationsb€twecnequilibrium thermodynamicdata
for reactants and products and kinetic parametersfor the enzyme-catalyzed proccssesinterconverting them are empirically
useful,surprising,and pres€ntlytheorctically unrationalized(at
least by us). It is tempting to consider cxplanations relating
differencesin stability betwcen reactantsand products to their
relative strength of binding to the enzyme,but we notc that valucs
of K^ and K, for theserelatively complcx enzymatic processcsdo
not necessarilycorrespond closely to physically interpretable
dissociationconstants. Further, it is unexpcctedthat free energy
relationship of the typcs obscrvedhere would hold through a series
of (apparentlyquite) different enzymes. It is possiblethat these
enzymes,as NAD(H)-depcndent alcohol dehydrogenases,
have
substantialsimilaritiesin their active sites. Alternatively,rhey
may sharesomedecpcrkinetic similaritiesreflectingthe fact that
all probably have a similar catalytic efficiency, in the sensediscusscdby Albery and Knowles.rt Thesemattersare of substantial
mechanisticinterest,but sincewe cannot prescntlydiscussthem
intelligently,and sincetheir theoreticalrationalizationis peripheral
( 3 8 ) A l b c r y , W . J . ; K n o w l c s J. . R . B i o c h e m i s t y 1 9 7 6 , 1 5 , 5 6 3 t - 5 6 4 0 .
-l
log x !q
-2
-3
-3-2-rOtZ3
log ( K,v Trgg / K 177a"g1agta
I
Figure7. Plotof log K'* vs.log (K,Rfo/K,oRcHoH;
for NAD(P)(H)dependent
dehydrogenases.
K'". is definedin eq 4. Dataare from Table
II: pointsare labcledwith numbcrscorresponding
to reactions
in Table
II.
to the central pointsof this paper,we will not discussthem further
theoretically.
Thc value of the correlation is summarized in Figure 6 and eq
5-7 to estimatethe value of K'* at which oxidations(which are
generallykinetically and thermodynamicallyunfavorable)become
kinetically favorable(K^l K, < 0.1) and thus suitablecandidates
for large-scalepreparativesynthesis. The answer,from eq 6, is
at app-roximatelyK'", = I X 1fr2. Reactionswith valuesof K,*
< l0-2 will procecdinefficiently while reactionswith K'* > l0-:
will be favorableenougbto accomplishsynthescsof *large' (> 100
mmol) quantitiesof substrate. By a similar analysis,reductions,
which are usually kinetically and thermodynamicallyfavorable.
becomekinetically unfavorablewhen K'*)
8 x l0{ and are
kinetically favorablcwhen K'* < 8 x l0-{.
Equation 5 suggeststhat the value of K^f K,. and thus the
efficiencyof enzyme-catalyzdreaction,dependsprimanly'on the
equilibriumconstantof the chemicaltransformationbeingconsideredand is not strongly influencedby the characteristicsof
7006 J. Am. Chem. Soc., Vol. 107, No. 24, 1985
the enzymecatalyst (within the seriesof oxidoreductases
consideredhere). This influenceof (or correlation between)K'* (or,
alternativelyK^l K) and reactionefficiencyis illustrated by the
HLADH-catalyzed oxidationof ethanolto acetaldehyde(without
removalof the product)-a reactionwhich is inefficientat any
concentration(K'* = 9 X l}-s: K^f K; = 3)-and the HLADHcatalyzedoxidationof cyclohexanolto cyclohexanon-a reaction
which proceedssmoothly(K; = 5 x l0-2: K^lKi = 0.04). An
exampleof a difficult reductionis the preparationof threo-socitrate, which has beenperformedin this group by two methods.6.le
In both cases.the thermodynamicsof the overall reactionwere
favorable;however,becauseof unfavorablekinetic parameters
( K ; = 1 . 3 M , K ^ I K ; = 1 3 0 ,K i = 1 . 3 x l 0 - { M ) , y i e l d sw e r e
poor and reactiontimes were long.
The useof NAD regenerationschemeswill be usefulonly with
preparativeenzymaticoxidationswhich have favorablekinetic
parameters(K./K1 < l). This category of reaction includes
HLADH oxidationsof diols to lactones,certain oxidationsgenerating stable ketonessuch as cyclohexanone,and the synthesis
of ribulose 1,5-diphosphate.If the problem of product inhibition
can be solved.other systemswill be practical, for instance,resolution of primary or sccondaryracemic alcoholsby enantioselectiveoxidation to the ketonesor aldehydes,or the preparation
of chiral a-hydroxy ketones or aldehydes by enantioselective
oxidation of the diols.
We emphasizethat the correlation in e.q3-5 describedabove
doesnot in any way replacethe task of finding the real value of
K; for a given K. and K'* but merely providesan "expectation
value- for the parameter. We bclieveeq 4 and 5 are a useful wav
to quantitate the enzymologist'sintuition when faced with the
possibilityof the occurrenceof product inhibition.
Experimentel Section
Gertenl. Enzymesand biochemicals
werepurchased
from Sigma.
cis-1,2-Cyclohexanedimethanol
(985o)waspurchascd
from Aldrichand
usedwithoutfurtherpurification.Methylenebluewaspurchased
from
Fisher.Hexaneand2,2.4-timethylpentane
wereFisherreagentgrade.
Water wasdoublydistilled,the secondtime througha Corningglass
AG-lb still. Oxygenwasrechnical
gradeandwasusedwithoutfurther
purification.GLC analysiswasperformedon a Perhn-Elmer3920Bgas
chromatograph
with a SE-30column.
Kinetics.Buffcredsolutionswerepreparedwith Hepes-KOH(0.1 M,
pH 7.6)or glycineKoH (0.1M, pH 9.0). n Perkin-Elmer
Model552
spectrophotometer
with thermctatedccll compartmcnt
(25 oC) wasused
for UV measurements
at 340,610,and665 nm. Thc slopcs,intercepts.
and errorsof the Eadie-Hofstecand pscudo.first-order
plots weredeterminedwith a least-squares
program.Stocksolutions
of mcthylene
bluc
andNADH weremadeaccordingto thcir nominalmolccularwcighsand
the final concentrations
determinedby mcasuringthc absorbances
of
dilutedsolutions
at 665nm (e 78000M-r cm-r)andat 340nm (c 5220
M-t cm-r), respcctivcly.
Rrtec of Uncrtdyzed Rcecdou bettccn Mcttylcrc Bhr rnd NADH.
Stocksolutionsof methylcncbluc (2.2 mM) and NADH (3.1 mM) in
waterwereprcpared.To a cuvcttc( 1.5mL) containingHcpcsbuffcr (0.1
M. pH 7.6,0.94mL) wercaddcdmcthylencblue(0.03mL. 0.066mM
in the cuvette)and NADH (0.02mL, 0.063mM in rhc cuvette).Thc
cuvettcwascappedand invcrtcdscveraltim6 to mix thc contcnts. Thc
rate of decreascin conccntration
of NADH wastakcn to bc the rate of
reactionbctweenmcthylcncbluc and NADH and wascalculatedfrom
thc initial rate of decrcasein abcorbancc
at 340 nm. Additionalrates
were measuredin a similar manner using approximatclycquimolar
conccntrations
of mcthylenebluc and NADH at oonccnrationsof 0.13.
0.16,0.19.and 0.26 mM. Two cuvetteswereprcparcdfor eachconccntration(a total of l0 rate measurements
weretaken)and an average
valucfor thc second-ordcr
rate constant,kr. wascalculatedto bc 3,2 I
0.2 M-' s-r. The proccdure
wasrepcarcdat pH 9 (0.1 M glycine-KOH);
the rate constantwasfoundto bc 5.3 + 0.5 M-t s-r lTable I).
Dctctuimtioo of Vducs of K, for Mcthylcm Bhrc for tbe t)lephonseCttrlyzcd Raction betrccn Mcttylc BhE rd NADH. Stock
solutions
of methylene
bluc (2.0 mM in water),NADH (2.5 mM in
watcr),and diaphorasc
(1.5 ml--rin Hepesbuffcr,pH 7.6) werepreparcd. To a cuvette(1.5 mL) containingHcpesbuffcr(0.1 M, pH 7.6.
0.90mL) wcreaddedmcthylcnc
blue(0.015mL,0.03 mM in the cu-
Lee and Ilhitestdes
v e t t e )a n d N A D H ( 0 . 0 6m L . 0 . 1 5 m M i n t h e c u v e t t e ) .T h e c u v e r r ew a s
c a p p e da n d i n v e r t e ds e v e r a lt i m e s t o m i x t h e c o n t e n t sa n d t h e r a t e o f
d e c r e a s ei n a b s o r b a n c a
e t 3 4 0 n m w a s m e a s u r e d( u r ) . D i a p h o r a s e( 0 . 0 2
m L , l 0 p g ) w a s a d d e da n d a s e c o n dr a r e w a s m e a s u r e d( t . ; ) . T h e d i f f e r e n c ei n t h e t w o r a t e s( r ' 2- u 1 ) w a s a t t r i b u t e dt o t h e d i a p h o r a s e - c a r a l y z e d r e a c t i o n . A d d i t r o n a ld i a p h o r a s e - c a t a l y z erda t e s w e r e m e a s u r e d
u s i n gt h e s a m ec o n c e n t r a t i o nosf N A D H a n d v a r v i n g c o n c e n t r a t i o nos f
m e t h y l e n eb l u e ( 0 . 0 4 , 0 . 0 6 . 0 . 1a, n d 0 . 2 m M ) . T w o c u v e t t e sw e r e p r e p a r e d f o r e a c hc o n c e n t r a t i o no f m e t h y l e n eb l u e ( a t o t a l o f l 0 m e a s u r e mentsof r'2- f 1 w€r€ made). The procedurewas repeatedat pH 9 0 (0 I
M g l y c i n e - K O H b u f f e r ) . E a d i e - H o f s t e ep l o t so f t h e d a t a a r e g r v e nr n
F i g u r e3 .
Determinrtion of the Vrluc of ff. for NADH in the Dirphorese.Crtelyzed Rerction of Methylene Blue end NADH. Stock solutrons o[
m e t h v l e n eb l u e ( 2 m M i n w a t e r ) . N A D H ( 0 , 9 3 m M i n w a r e r ) .a n d
d i a p h o r a s e( 1 . 5 m g i n l 6 m L o f p H 7 . 6 H e p e sb u f f e r ) w e r e p r e p a r e d
T o a c u v e t t e( 1 . 5 m L ) c o n t a i n i n gg l y c i n e - K O H b u f f e r ( 0 . 1 M . p H 9 0 .
0 . 7 1m L ) w a sa d d e dm e t h y l e n e
b l u e( 0 . 2 5m L , 0 . 0 2 m M i n r h e c u v e r t e )
T h e c u v e t t ew a s c a p p c da n d i n v e r t e ds e v e r a lt i m e s t o m i x t h e c o n r e n t s
a n d t h e r a t e o f d e c r e a s ei n a b s o r b a n c ea t 3 4 0 n m w a s m e a s u r e d( r . ,)
Diaphorase(0.02 mL, 2 pg) was added and a secondrate was measureo
( u z ) . T h e d i f f e r e n c ei n t h e t w o r a t e s ( u 2 - u , ) w a s a t t r i b u t e d t o t h e
diaphorase-catalyzedreaction. Additional diaphorase-catalyzedra tes
were measurcd in a similar manner using the same concentratronof
m e t h y l e n eb l u e a n d v a r y i n g t h c N A D H c o n c e n r r a t i o n( 0 . 0 3 ,0 . 0 6 , 0 . 1 8
mM). No differencc in diaphorase-catalyzed
rate with varying NADH
concentration(uz - u,) was observedat theseconcentrationsof NADH;
t h e v a l u e o f K , o f o r N A D H w a s a s s u m e dt o b e < l x l 0 - 5 M . A n e x a c r
value for the value of K, for NADH was not measured.
Rete of Reaction betweenReducedMethyhne Blue end Dioxygen. A
stock solutionof reduccdmethyleneblue was preparedby mixing a dark
blue, argon-prrgcdsolutionof methyleneblue (2.0 mM, 8 mL, l6 smol).
N A D H ( l l m g , l 3 r r m o l ) .a n d d i a p h o r a s e( 2 m g ) . T h e c o l o r o f t h e
solutron changed from deep blue to light blue. The concentrarronof
r e d u c e dm e t h y l e n eb l u e w a s a p p r o x i m a t e l y1 . 6 m M ( s e e b e l o w ) . A
c u v e t t e( 1 . 5 m L ) c o n t a i n i n gH e p e sb u f e r ( 0 . 1 M , p H 1 . 6 . 0 . 9 ' 7m L ) w a s
€pped with a rubber s€ptum. Inlct and ourlet necdleswere artached and
oxygenwas bubblcd through the solutionfor 20 min. An aliquot (i0 rrL)
of the reduced methylene bluc solution was transferred to the cuverte
using a l0GrrL gas-tightsyringe. The initial absorbanceat 610 nm (the
e x t i n c t i o nc o e f f i c i e n t 1
, 5 , 1fso, r m e t h y l e n eb l u c i s 4 4 5 0 0 M - r c m - r ) w a s
measured(OD = 0.5) and the conccntrationof unreactedmethyleneblue
in the stock solution was calculatcd ro bc 0.4 mM. (The UV specrrum
of methylenc blue has a sccond,smaller absorbancemaximum at 610 nm:
this wavelengthwas uscd so that a wider conccntrationrange could be
examined.) The diffcrencc bctwecn the initial and final concentrarrons
of metbylcnc bluc in thc stock solution was assumedto be rhe concentration of reduccdmethylenebluc. The absorbanceat 610 nm was read
dircctly from thc spcctrophotometer every 30 s for 3 min. The proccdure
was repeatedusing glycineKOH buffcr (0.1 M, pH 9). A plot of the
data is given in Figurc 2.
Rrtes of Rercdor
bctrcen NADH end Othcr Elcctron-Tnnsfer
Rergents Uocrtdyzed end Crtelyzed Rrtes. Thc uncatalyzed rates of
reaction betwecn NADH and various electron transfcr reagents(methyl
viologen,flavin mononucleotide,dichloroindophenol,phenazinemetho.
sulfate, and potassiumfcrricyanide) werc measuredat pH 7.6. Stock
solutionsof the elcctron-transfer rcagent ( l0 mM in water), NADH ( l2
mM in water), and diaphorasc(l mg/ml in Hepes buffer of pH 7.6)
were preparcd. To cuvettc (1.5 mL) containing Hepcs buffcr (0.1 M.
pH 7.6,0.94 mL) wcrc addcd NADH (0.02 mL,0.24 mM in the cuvette)
and electron-transferreagent (0,02 mL. 0.02 mM in the cuvette). The
solutions in the cuvcttcs wcre mixcd and thc rate of decreasein absorbanceat 340 nm was measurcd(ur). For all thc electron-transferreagents except for phcnazinc mcthculfate, the decreascin absorbanceat 34O
nm \ryasattribut€d to thc dccreasc in conccntration of NADH alone. and
thc extinction cocfficient for NADH (e = 6220 M-r cm-r) was used ro
calculatc thc rate. Thc oxidizcd form of phenazinc mcthosulfatc has a
high cxtinction cocfficient at 340 nm (c = 2600 M-r cm-r); the sum of
thc two extinction cocfficicns was uscd to calculatc thc rate. Diaphorasc
(0.02 mL, 20 p0 was addcd and a second ratc mcasured (u2). The
differencc in thc two rat6 (u2 - u1) was attributcd to diaphorasc-catzlyzd
reaction. Thc data for both the uncatalyzcd and catalyzd rares are given
in Table I.
Enzym Ascrys. Assayswere performcd using literature procedures.rB
U n i t s o f e n z y m a t i ca c t r v i t ya r e r J m o lm i n - r . U n i t s o f H L A D H r e f e r t o
activity with cis-cyclohcxancdimethanol
as the substrare(the value of
V^u for cis-cyclohexanedimethanol
is approximately 80Voof the value
of V*, for cyclohexanolra). Diaphorasewas assayedas describedfor rhe
kinetic determinations,above,except with methylene blue ar a concent r a t i o n o f 0 . 2 m M i n t h e c u v e t t e . A s s a v so f H L A D H a n d e l u c o s ed e -
In Situ Regenerationof ,\AD from NADH
I
24. t985 7007
,3'
m
lr6
1
"
x
o
t
e
o
lrO
a
O18
o
l.z
(mM-lmrn)
t
o
o
o
o
t
.
;--
t
0
o
t
o
,N.
Or6
0.4
o
tal
o
o
a
ct
o
Q.2
I
o
O.O
, (\,t,
0204060
Timc(h)
'lc?
-O.8
-Oo4
O
Or4
t!
O.8
lo2
116
2rO
(mM
)
Figure t. Dixon plot used in the determination of the value of K, for
acetaldehydein the HLADH-catalyzeA oxidations of cis-cyclohexancdimethanol (O) and ethanol (t). The value of K, was I mM for both
reactions. The assayswere performed on different days with different
solutionsof enzyme: the relative values of VM, for the two substrates
shown here may not bc accurate.
0.6
I
v
0.4
(mM'rmrn)
-rc
-40
-20
0
20
I
40
60
80
too
(mM)
Figure 9. Dixon plot used in the derermination of the value of K1 for
cyclohexanonein the HLADH-catalyzed oxidationof cyclohexanol.The
v a l u eo f K , w a s 4 0 m M .
hydrogenase(GlcDH) with added Na2SOaor NaCl were accomplished
by varying the concentrarionof added salt between 0 and 0.6 M for
N a 2 S O aa n d b e t w e e n0 a n d 1 . 2 M f o r N a C l a n d a s s a y i n ga s u s u a l .
Determinrtion of I(. The value of K, of acctaldchyde was determined
f o r t h e H L A D H - c a t a l y z e d o x i d a t i o n so f e t h a n o l a n d c i s - 1 , 2 - c y c l o h e x a n e d i m e t h a n oal t p H 9 . 0 ( g l y c i n e - K O H ) . F i g u r e 8 s h o w s D i x o n
p l o t so f t h e d a t a . T h e c o n c e n t r a t i o n o
s f e t h a n o la n d N A D w e r e m a i n tainedat 30 mM (60 times the value of K, for ethanol;secTablc II) and
I mM (40 times K.), respectively,while thc conccntration of acetaldehyde was varied between0 and 2 mM. Thc concentrationsof cyclohexanedimethanol
and NAD were maintainedat 100 mM (4K* close
to its solubility limit) and 2 mM (40Km), respcctivcly,while the conccntrationof acetaldehydewas vaned between0 and Lt mM. The value
of K1 was I mM for both reactions.
The value of ,(i of cyclohexanoncfor the HlADH-catalyzed oxidation of cvclohexanolwas detcrmincd similarly (Figurc 9).
Enzyme Immobilizetion. HLADH and diaphoras€ were immobilized
on polyacrylamidegel as previouslydcscribcd.r HLADH was immobilized in lV3lVa yield. and diaphorasein 30-.4O%yicld. Aftcr thc gcls
had been stirred in a reactor for scveraldays, the activity of the recovered
enzyme was often highcr than the initial activity. l'-his increascin activity
is due to the additional grinding rhe gel receivesundcr reaction conditions. This grinding reducesthe size of the gcl particles and decreascs
rate limitations due to slow diffusion of substratesinto the intcrior of
large particles.
Oxidrtion of cis-1,2-Cyclohcxencdimethrnot Using HLADH snd
GIDH. A 3-L, three-necked,round-bottomed flask equippcd with a
m a g n e t i cs t i r r i n g b a r w a s c h a r g c dw i t h 2 - o x o g l u t a r i ca c i d ( 2 2 . 4 g , 1 5 3
m m o l ) a n d w a t e r t 0 . 7 5 L ) . A m m o n i u m h y d r o x i d e( 3 0 V o , 3 5m L ) w a s
a d d e d t o n e u t r a l i z et h e a c i d a n d t o a d j u s t t h c p H t o 8 . 1 . c i s - 1 . 2 C y c l o h e x a n e d i m e t h a n( ol 0l g . 6 9 m m o l ) . s o l u b l eH L A D H ( 2 5 0 m g , 5 8 0
U ) . a n d i m m o b i l i z e dC I D H ( 2 3 0 U . 1 0 0 m L o f g e l ) w e r e a d d e d . A n
a l i q u o t( 0 . 5 m L ) o f t h e m i x t u r ew a s r e m o v e d .N A D ( 0 . 2 09 , 0 . 2 7 m m o l )
a n d h e x a n e( l L ) w e r e a d d e d . T h e m i x t u r e w a s s t i r r e d o n l y r a p i d t y
e n o u g h t o s u s p e n dt h e g e l i n t h e a q u e o u sp h a s e , T h e e x r e n r o f r h e
r e a c t i o nw a s m e a s u r e db y r e m o v i n ga l i q u o t s ( 0 . 5 m L ) o f t h e a q u e o u s
Figure 10. Plot of the progressof an oxidation catalyzed by HLADH
o f c i s - l , i - c y c l o h e x a n e d i m e t h a n oR
l . e g e n e r a t i o no f N A D w a s a c c o m plished using 2-oxoglutarate/GlDH: the reacrionwas followed by periodic assaysof residual 2-oxoglutarate. The subscriptsr and 0 on the
nght axis refer to conc€ntrationsof 2-oxoglutarateat time t after the start
of the reaction and at time = 0.
phase,centrifuging to separatethe gel, diluting a portion of the supcrn a t a n t i n a l : 5 0 r a t i o , a n d q u a n t i t a t i v e l ym e a s u r i n g2 - o x o g l u t a r a t eb y
enzymatic assay. Figure l0 shows a plot summarizing the extent of
reaction vs. time. An accurate determination of the volume of the
aqueousphasewas found by enzymatic assayof 2-oxoglutararein the
a l i q u o t r e m o v e da t t h e s t a r t o f t h e r e a c t i o n ;t h e k n o w n ,i n i t i a l q u a n t i r y
of i53 mmol of 2-oxoglutaratewas usedin the calculationand a reacrion
v o l u m e o f 1 . 0 5 L w a s o b t a i n e d . A c c u r a c y i n t h e m e a s u r e m e not f t h e
volume was ne@ssaryfor the subsequent,quantitativedeterminationsof
r e s i d u a l2 - o x o g l u t a r a t e .
T h e r e a c t i o nw a s 9 4 V oc o m p l e t ea f t e r 2 . 5 d a y s . E n z y m a t i ca s s a yo f
t h e r e a c t i o nm i x t u r e s h o w e dt h a t 0 . 1 7 m m o l o f N A D ( 6 0 V o )a n d 0 . 0
m m o l o f N A D H r e m a i n e d . T h e t u r n o v e rn u m b e r ( T N , e q u i vo i p r o d u c t / e q u i v o f N A D ) f o r N A D w a s 5 0 0 . T h e h e x a n el a v e r w a s r e m o v e d
by iorccd siphon using a stainlesssteelcannula and concentratedto a pale
. v " e l l oowi l ( 6 . 3 g ) . T h e e n z y m e - c o n t a i n i ngge l w a s a l l o w e dr o s e t t l ea n d
t h e a q u e o u ss u p € r n a t a n tr e m o v e dv i a c a n n u l a . T h e r e m a i n i n gg e l w a s
assayedand found to contain45 U (20Eo)of residualGIDH activitv. The
a q u e o u ss o l u t i o nw a s c o n c e n t r a t e tdo 0 . 2 5 L b y b a t c h w i s eu l t r a f i l t r a t i o n
a t 4 0 p s i u s i n ga s t i r r e d A m i c o n c e l l ( 3 5 0 m L ) a n d a D i a f l o m e m b r a n e
w r t h a 1 0 O 0 O - d a l t ocnu t o l f . T h e r e s i d u a tH L A D H a c t i v i t l w a s f o u n d
to bc 140 U (24Eal. The aqueoussolurronwas acidified with HCI to pH
- 5 a n d e x t r a c t e dw i t h t h r e e 1 5 0 - m L p o r t i o n so f e t h e r . T h e e t h e r e a l
solutionwas dned (MgSOr) and concrnrrard to a yellowoil (3 0 g). The
c o m b i n e d y i e l d o f t h e c r u d e l a c t o n e w a s 9 . 3 g ( . 9 6 E a ) .T h e o i l w a s
d i s t i l l e dt h r o u g h a v a c u u m - j a c k e t e sdh o r t - p a t hd i s t i l l a t i o nh e a dt o g i v e
( + ) - ( I R , 6 S ) - c r s - 8 - o x a b i c y c l o [ 4 . 3 . 0 ] n o n a n - 7a- soan ec o l o r l e s so i l w h i c h
s o l i d i f i e du p o n s t a n d i n g a t r o o m t e m p € r a r u r e( 8 2 g , 8 5 7 0y i e l d ) : b p
6 4 - ' 7 4" C ( 0 . 2 t o r r ) ( l i t . r ab p 8 6 o C ( 2 t o r r ) ) : a 2 3 o+ 4 3 . 8 o ( n e a t ) . I a ] : r e
+ 4 8 . 8 o ( c 0 . 5 g l l 0 0 c m r , C H C I 3 ) ( l i t . r a[ a ] 2 5 p+ 4 8 . 8 o( c 0 . 5 g 7 1 0 0
c m l ) ) ; l H N M R d a t a w e r e i n a g r e e m e n tw i r h l i t e r a t u r ev a l u e s .
Oxidetion of cis-I,2-CyclohcxenedimethenolUsing HLADH and
MB/Diephorrse. To a l-L round-bottomed flask equipped with a
m a g n e t i cs t i r r i n g b a r w e r e a d d e d H e p e sb u f f e r ( 0 . 1 M , p H 7 . 6 , 0 . 1 L ) ,
c i s - c y c l o h e x a n e d i m e t h a(n4ogl , 2 8 m m o l ) , N A D ( 0 I g , 0 . 1 3 m m o l ) ,
m e t h y l e n eb l u e ( 3 0 m g , 0 . 0 8 m m o l ) . i m m o b i l i z e dH L A D H ( 2 0 U . 9 0
m L g e l ) . i m m o b i l i z e dd i a p h o r a s e( 2 7 [ J , 4 0 m L o f g e l ) , a n d s o l u b l e
c a t a l a s e( 1 0 m g ) . T h e s o l u t i o nt u r n e d f r o m d e e pb l u e t o c o l o r l e s sa f t e r
2 . 0 m L . u s e da s
2 0 m i n . H e x a n e( 5 0 0 m L ) a n d 2 , 2 , 4 - t r i m e t h y l h e x a n( e
an internal GLC standard) were added. Oxygen was bubbled through
the aqueous solutions via a stainlesssteel needle. The reaction was
monitored by GLC analysis of the organic phasc. After 4 days, the
hexane layer was decantedand the aqueousphasecentrifuged to remove
t h e g e l . T h c g c l w a s w a s h e dw i t h a d d i t i o n a lb u f f e r ( 1 0 0 m L ) a n d t h e
combined aqueoussolutionswere acidified to pH 3 and extracted with
hexane(2 x 0.1 L). Thc combincdorganic solutionswere concenrrared.
redissolvedin ether (20 mL), filtcred through decolorizingcarbon (0.5
g ) , a n d c o n c e n t r a t e tdo a c o l o r l e s so i l ( 3 . 2 g , 8 3 V o ) . T h e r H N M R
s p e c r r a ld a t a a g r e e dw i t h l i t e r a t u r ev a l u e s : [ o l 2 ' o * 4 8 o ( c 0 . 5 g / 1 0 0
c m r . C H C I j ) ; l i t . [ a l 2 5 e+ 4 8 . 8 0 . T h e r e s i d u a el n z y m ea c t i v i r i e sw e r e :
H L A D H . 3 0 U , 1 6 0 7 od; i a p h o r a s e , 8 U
5 , 3 0 0 E o .T h e a q u e o u s o l u r i o n
w a s a s s a y e df o r r e s i d u a lN A D a c t i v i t y( 0 . 0 4 m m o l . 2 8 q r ) . T h e r u r n o v e r
n u m b e r f o r N A D w a s 3 5 0 :t h c t u r n o v e rn u m b e rf o r m e t h y l e n eb l u e w a s
580
Oxidetion of GlucoseUsing GIcDH end Fe(CN)ur-/Diephorese. ,{
5 t l 0 - m L r o u n d - b o t t o m efdl a s ke q u i p p e dw i t h a m a g n e t i cs r i r r i n gb a r w a s
c h a r g e dw i t h g l u c o s et 0 9 g , 5 m m o l ) .N A D ( 0 . 0 19 . 0 . 0 4 m m o l ) .i m m o b r l i z e dG l c D H . a n d d i a p h o r a s e( 4 4 a n d 2 2 U , r e s p e c t i v e l yS. 0 m L o f
7008
g e l ) a n d H e p c sb u f f e r ( 0 I M . p H u . 6 . 7 0 m L ) . p o t a s s i u m
ferncvanrde
( 3 I g ' l 0 m m o l ) w a s a d d e da n d t h e m i x t u r e w a s s r i r r e d
u n d e ra n a r g o n
a t m c p h e r e . T h e p H w a s m a i n t a i n e da t 7 . 0 b y a u r o m a r i ca d d i t r o no f
2.5
N K o H . A l i q u o t s ( 0 . 5 m L ) o f r h e r e a c r i o nw e r e r e m o v e dp er i o d i c a i l y
a n d c e n t r i f u g e da. p o r t i o no f t h e s u p € r n a t a nw
t a sd i l u t e dr n a I : 2 0 r a t i o .
a n d t h e a b s o r b a n c ew a s m e a s u r e da t 4 2 0 n m . A f t e r 6 h . 6 3 m L o f 2 . 5
N K o H ( 1 5 . 8m m o l , J . 2 e q u i v )h a d b e c na d d e d . F i g u r e4 s h o w s p r o t
a
o f t h e a d d i t i o no f b a s eu s . t i m e f o r t h e r e a c r o ra n d a p l o t o i t h e r a t i o
of
t h e a b s o r b a n c et o i n i t i a l a b s o r b a n c ea t 4 2 0 v s . t i m e . T h e m i x t u r e
was
centrifugedto separatethe gel. The activititesof the recovered
enzvmes
w e r e 6 0 U f o r G I c D H ( l . t 0 q o )a n d i 2 U i o r d i a p h o r a s e
il50s").-Th;
t u r n o v e rn u m b e r f o r \ A D w a s 1 2 5 . T h e p r o d u c t o f t h e
oxrdation.
g l u c o n a t ew
. asnot isolated.
D e t e r m i m t i o n o f p e r t i t i o n C o e f f i c i e n t so f ( + ) _ ( l R . 2 S ) - c i s - t - O x r _
bicyclof4.3.0frcnan-7-onebetreen Hexrne end wrter. A
solution oi the
l - a c t o n(e5 5 m g , 0 . 3 9 m m o l , 7 8 m M ) a n d o c t a d e c a n (cj 0 m g . 0 . l 2
mmol.
2a mM) in hexane(5 mL) was analyzedby GLC, ( 107"CaiLwai).
n"
t::pol.., factor.
*"t found to be I 9l (R/ =
[mmol of lactone/mmol
.fr.
olr octadecanelllareaof lactone/areaof ociadecanel). {
l-mL portion
o f t h e h e x a n es o l u t i o nw a s e q u i l i b r a t e db y s h a k i n gw i t h a 2 - m L
aliquot
each of glycine buffer (0.25 M. pH 9.0) and oidistiiled water.
The
h e x a n el a y e r w a s a n a l y z e db y G L C u s i n g o c t a d e c a n ea s t h e
internal
standard. The partition coefficients(o) wifh glycine buffcr and Jistilled
w a t e r w e r e 1 . 0 a n d 1 . 9 ,r e s p c c t i v e l y ,
tlecompc{tion of oxrloacetrte
A stock solution of oxaloacctate ( ltn
mM in H"po buffer, pH 7.6) was preparedby combiningoxaracctrc
aod
( 1 3 2 m g . I m m o l ) . l ' . ! a O H( 1 . 8 m L o i a I \ s r a n d a r d
s o l u r r o n r. .i n o
H e p e sb u f f e r ( E . 2 m L ) . T h r e e v i a l s ( 1 0 m L ) w e r e p r e p a r e d :r h e
iirsr
c o n t a i n e dH e p e s( 0 . 1 M ) , g l y c i n e( 0 . 1 \ { . p H 7 . 6 ,+ . S ' n r L ta. n d
orala c e t a t e1 0 . 5m L ) : t h e s e c o n dc o n t a i n e dH L A D H ( 5 m g y .H e p e s( 0
I V.
p H 7 . 6 . 0 . 9m L ) . a n d o x a r a c e r a (r 0
e r m L ) ; t h e t h i r dc o n t a i n e d
Hepes
( 0 . 1 M , p H 7 . 6 . 4 . 5m L ) a n d o x a l a c e t a (t 0
e . 5m L ) . A l i q u o t sr 5 0 s L )
w e r e r e m o v e da n d a s s a y e df o r o x a l a c e t a t e . r tF i g u r e I s h o w s
a plor oi
t h e f i r s t - o r d e rd e c o m p o s r t i o n .
Acknowledgment. This work was supported by the Natronal
_
I n s t i t u t e s o f H e a l t h , G r a n t G M 3 0 3 6 7 . C i u r c o l l e a g u e sp r o f e s s o r
Jeremy Knowles and Steve Benner offered usefur iuggestions
on
several aspects of this work.
R e E s t r yN o . I , 15 7 5 3 - 5 0 - l ;2 , 6 5 1 7 6 - 0 2 - 5N; A D . 5 l _ 8 ; l _ 9\;A D H .
5 8 - 6 8 - 4M
; B . 6 1 - 7 3 - 4M
: V . l 9 l 0 - 4 2 - 5 :F M N . 1 4 6 - t 7 - 8 D
; Clp. e56a 8 - 9 ;P M S , 2 9 9 - ll - 6 ; O ? , i i 8 2 - 1 + 7 ; F e ( C N ) o F .1 3 4 0 8 _ 6 2 -Ci :H , C H O .
7 5 - 0 7 - 0 ; E I O H , 6 4 -t 7 - 5 ; K 3 F e l C N ) u , t 3 i 1 6 - 6 6 - 2 : g l u t a m a i e
de_
h y d r o g e n a s e9, 0 2 9 - 12 - 3 ; d i a p h o r a s c9, 0 0l - I g - 7 ; o - g l u c o l e ,5 0 - 9 9 - 7
: ogluconate, 526-95-4: alcohol dehydrogenase,903 l-lZ-S: glucose
deh y d r o g e n a s e 9. 0 2 8 - 5 3 - 9 ;o x i d o r e d u c t a s e9. 0 5 5 -I 5 - 6 : d e h i d r o g e n a s e .
9 0 3 5 - 8 2 - 9 :c y c l o h e x a n o n e1, 0 8 - 9 4 - r; c y c r o h e x a n o rr, 0 g - 9 3 - 0 ; ' m a r a t e
d e h y d r o g e n a s e9,0 0l - 6 4 - 3 ;g l y c e r o rd e h y d r o g e n a s e9,0 2 g -l 4 - 2 . i a c t a r e
dehydrogenase,
900l -60-9,:malic enz),me,g}tg-4l -l ; 3-hydroxrbutr rare
d e h y d r o g e n a s e9 ,0 2 E - 3 E - 0i ;s o c i t r a t ed e h y d r o g e n a s e9,0 0t - 5g _5: l - o x o 8lutarate.328-50-7.