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Cell Death and Differentiation (1999) 6, 1054 ± 1059
1999 Stockton Press All rights reserved 13509047/99 $15.00
http://www.stockton-press.co.uk/cdd
Review
Catalytic properties of the caspases
HR Stennicke1 and GS Salvesen*,1
1
The Program for Apoptosis and Cell Death Research, The Burnham Institute,
10901 North Torrey Pines Road, La Jolla, California, CA92037, USA
* Corresponding author: GS Salvesen, The Program for Apoptosis and Cell
Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La
Jolla, California, CA 92037, USA. Tel: +1 (619) 646 3114;
Fax: +1 (619) 646 3189; E-mail: [email protected]
Received 20.5.99; accepted 21.9.99
Edited by D Nicholson
Abstract
Caspase stands for cysteine-dependent aspartate specific
protease, and is a term coined to define proteases related to
interleukin 1b converting enzyme and CED-3.1 Thus their
enzymatic properties are governed by a dominant specificity
for substrates containing Asp, and by the use of a Cys sidechain for catalyzing peptide bond cleavage. The use of a Cys
side chain as a nucleophile during peptide bond hydrolysis is
common to several protease families. However, the primary
specificity for Asp turns out to be very rare among protease
families throughout biotic kingdoms. Of all known mammalian
proteases only the caspase activator granzyme B, a serine
protease, has the same primary specificity. In addition to this
unusual primary specificity, caspases are remarkable in that
certain of their zymogens have intrinsic proteolytic activity.
This latter property is essential to trigger the proteolytic
pathways that lead to apoptosis. Here we review the known
enzymatic properties of the caspases and their zymogens
within the broad context of structure:mechanism:activity
relationships of proteases in general.
Keywords: protease; proteolysis; catalytic mechanism; caspase
Abbreviations: AMC, 7-amino-ymethyl-coumarin; +PA, tissue
plasminogen activator
Breaking the peptide bond
The emergence of amino acids as the building blocks of
proteins allows for the combination of stability, strength, and
structural features unique to the peptide bond. However, this
strength and stability of the peptide bond also makes specific
cleavage or degradation of proteins by hydrolysis much more
than a simple `just add water' problem. This is evident from the
fact that non-catalyzed hydrolysis of proteins requires
prolonged heating even in the presence of strong acids.
Thus, enzymes that cleave peptide bonds (peptidases or
proteases) have high energy barriers to overcome. The term
protease is synonymous with peptidase, meaning peptide
bond hydrolase, and includes endopeptidases and exopeptidases. Caspases are strict endopeptidases. From a mechanistic point of view most proteases utilize their ability to force the
trigonal planar peptide bond into a tetrahedal geometry as a
prerequisite for hydrolysis. Thus, the majority of the available
binding energy is used for stabilizing this tetrahedral
intermediate rather than merely forming an enzyme-substrate
complex. The ability to catalyze the hydrolysis of the peptide
bond at neutral pH and ambient temperatures therefore
characterizes proteases, where various catalytic mechanisms
are located in a variety of otherwise unrelated protein scaffolds.
At least five distinct catalytic mechanisms have been
discovered in proteases, but irrespective of the type of
residues involved in the particular mechanisms, the key
features are identical. About the point in the reaction
coordinate where the enzyme pulls the peptide bond into
tetrahedral geometry, a nucleophile adds to the carbonyl
carbon of the scissile bond. In some families of proteases (the
metallo- and aspartic protease classes) this is simply done by
making a water molecule nucleophilic through specific
interactions to groups in the protease. In others (the
cysteine, serine, and threonine protease classes) a sidechain within the protease acts as the nucleophile, forming a
covalent adduct (an ester) with the substrate during catalysis.
Hydrolysis of substrate by these latter classes occurs by
displacement of the ester. The second step in peptide bond
hydrolysis is the protonation of the a-amino moiety of the
leaving group, which is particularly important in cysteine
proteases such as the caspases due to the lower stability of
the thiol ester intermediate compared to regular esters.
In the case of the caspases it is the formation of a
tetrahedral intermediate by promotion of a cysteine residue
to act as a nucleophile that is the pivotal point in catalysis
and thus, we will focus on this particular mechanism for
promoting hydrolysis. The organization of the catalytic site,
the geometry of the residues, and the distance between
them, all have a variety of implications for protease
functions in vivo. This particular point becomes very clear
when comparing two proteases that uses the same
catalytic machinery to promote the breakage of the peptide
bond, but on quite different scaffolds as it is the case for
the caspase family and the papain family. Though they use
essentially the same catalytic mechanism, these two
families of cysteine proteases have some very significant
differences in the promotion of catalysis as well as the
organization of the substrate binding site.
Catalytic mechanism
Like other cysteine proteases, the caspases contain a
catalytic Cys-His pair with Cys285 acting as the nucleophile
and His237 acting as the general base to abstract the proton
from the catalytic Cys and promote the nucleophile (Figure 1).
The numbering of caspase residues follows the caspase 1
Catalytic properties of the caspases
HR Stennicke and GS Salvesen
1055
convention. Interestingly, serine proteases (such as trypsin,
tissue plasminogen activator, and subtilisin) also frequently
contain a His residue that accompanies the catalytic Ser. In
the model cysteine protease papain, where the cysteine
protease catalytic mechanism has been studied most
extensively, it is believed that the Cys-His dyad exists as an
ion-pair where the thiol proton of the catalytic Cys has been
transferred to the acceptor His before substrate binding.2 Prepolarization of the catalytic nucleophile represents one of the
major differences between the serine and cysteine proteases,
since in the serine proteases the nucleophile is believed to
develop along the reaction coordinate. While the ability of the
catalytic His to abstract a proton from the catalytic Cys is
extremely important, the ability to protonate the a-amino group
of the scissile bond is expected to be at least as important in
the caspases. The reason for the importance of the
protonation of this group lies in the properties and reactivity
of the thiol ester: its susceptibility to nucleophilic attack. Thus,
if the a-amino group is not protonated the reformation of the
peptide bond will be favored over the release of the leaving
group, and consequently proteolysis will not occur.
In many proteases the side-chain of a third residue plays a
significant role in the promotion of catalysis. The majority of
the members of the papain family contain an Asn side-chain
that is believed to orient the histidine in the Cys-His ion-pair,
thereby influencing catalysis.3 Indeed, when Asn175 is
replaced by an alanine in papain, the catalytic activity is
decreased by more than 100-fold mainly due to a significant
drop in kcat.3 In the caspases this third component of the
catalytic triad is not a side-chain, but rather is proposed to be
the backbone carbonyl group of residue 177, which in
caspase 1 is a proline and in caspase 3 a threonine.4,5
Unfortunately, the importance of this third member is difficult
to establish in the caspases since the interactions are to a
backbone moiety, which cannot easily be experimentally
verified by mutagenesis. Furthermore, alignment of the
known mammalian caspases (Accession PF00656)6 does
not reveal any significant conservation in the region
surrounding the putative third member of the triad, allowing
us to raise the hypothesis that the putative third member may
not exist in the caspase structure and mechanism.
Investigations of the pH dependence of caspase
catalysis have provided useful insights into the catalytic
mechanism. Human caspases exhibit only minor differences in pH profiles, and all are maximally active within the
pH range and ionic strength of cell cytosols.7,8 They all
exhibit a rather narrow bell-shaped pH dependence with
optima in the range 6.8 ± 7.2 signifying the existence of one
active form of the enzyme with the increase in activity most
likely due to the de-protonation of the catalytic Cys residue.
In this respect the caspases closely resemble other
unrelated cysteine proteases such as papain in their
activity pH profiles although the pH-profile is much more
narrow than that found for papain.9 Interestingly, the
substitution of the third member of the catalytic triad in
papain also results in a considerable narrowing of the pH
profile due to the increased distance between the catalytic
residues.3 This observation supports the lack of a third
member of the catalytic triad in caspases since examination
of the structure of papain complexed with leupeptin reveals
a distance between the catalytic residues to be 3.75 AÊ,10
which is significantly less than the distance of 5.2 AÊ found
in the caspase:aldehyde structures.4,5 Interestingly, the
large distance between catalytic Cys and His residues in
caspase structures argues against an ion pair, with the
implication that the Cys may not be pre-polarized.
The substrate binding site
Figure 1 The minimal caspase catalytic site. The role of the catalytic site of
proteases may be summarized as the ability to force formation of a tetrahedral
intermediate, and the following explanation derives from the 3-D structures of
caspases 1 and 3.4,5 The substrate chain (white text) lies in the binding site
with the scissile bond positioned close to the catalytic residues. The backbone
amides of Gly238 and Cys285 (which constitute the `oxyanion hole' in elliptical
shading) donate H-bonds to the carbonyl oxygen, thus polarizing the carbonyl
group of the scissile bond. The carbon is now electrophilic and susceptible to
attack by the juxtaposed nucleophilic thiol of the catalytic Cys285 (triangle
shading). Prior to or during the nucleophilic attack on the carbonyl carbon the
thiol group of Cys285 donates its proton to His237 (square shading), which
then can act as the catalytic acid by protonating the a-amino group of the P1'
amino acid. During deacylation of the enzyme His237 will help to polarize the
water molecule required for completing the hydrolysis by forming the second
tetrahedral intermediate
The substrate binding site in all proteases is composed of a
fairly large number of amino acid residues that secure proper
alignment of the substrates prior to hydrolysis and help
promote catalysis through stabilization of the transition state.
The binding site is divided into a number of sub-sites (see
Figure 2), each securing a single amino acid residue of the
substrate by multiple interactions. In addition to interactions
with specific side-chains, binding of the peptide backbone
also plays an important role in catalysis.
The substrate specificity of the caspases will be
described elsewhere in this issue and we will cover the
essential details briefly for comparative purposes. Although
no detailed studies have been done to address the P1
specificity since the original description of caspase 1,11 all
the caspases are believed to possess a strict preference for
Asp in the enzyme S1 pocket as previously indicated in the
origin of the family name. In the papain family the primary
specificity pocket is S2, which tends to prefer hydrophobic
substrate side-chains. Indeed, there is no detectable S1
Catalytic properties of the caspases
HR Stennicke and GS Salvesen
1056
S2
S4
S3
S1’
S1
S2’
Figure 2 The protease substrate binding site. The peptide chain of a
substrate (or inhibitor) lies across the substrate-binding cleft of the protease.
The side-chains of each residue of the substrate are numbered sequentially,
with the `P' designation, from the scissile bond (dashed). By convention, 59
those towards the N-terminal of the substrate are called the `unprimed' side,
and those toward the C-terminal of the substrate are called the `primed' side.
Complementary pockets on the surface of the protease are given the `S'
designation. Though the P sites constitute a single side-chain, the S sites are
each usually composed of several different portions of the enzyme, since they
define a binding surface. Though this convention is extremely useful for
understanding protease specificity, it is important to remember that not all
subsites are occupied in different proteases, and subsites are not always truly
independent60
preference throughout the papain family,12,13 and the
substrate backbone is even bound in a different way than
in the caspases.10
When compared to the papain family of proteases, this
way of accommodating the P 1 residue provides an
additional explanation for the drop in activity observed in
the acidic pH range. This is because protonation of the side
chain carboxylate of the P1 Asp residue would make it
unable to bind to the basic S1 subsite of the caspases. That
only the negatively charged form of the P1 Asp residue will
result in productive binding of the substrate is supported by:
(i) the strict preference for Asp in this position as compared
to Asn, and (ii) the close similarity in the pH dependence in
the low pH range between the caspases and other
proteases with preferences for negatively charged P1
substituents (glutamyl endopeptidases from Streptomyces
griseus14 and Staphylococcus aureus V815).
The other significant feature of enzyme substrate
interactions is the influence of peptide backbones on
substrate/inhibitor binding. Although the importance of
these backbone interactions is difficult to investigate in
detail due to the problems associated with changing the
structure of the backbone, this is a central part of the
binding of the substrate during catalysis. In all the caspase
structures the backbone of the bound inhibitor forms an
anti-parallel b-sheet like structure with the enzyme.4,5
Similar binding modes are found for the chymotrypsin
family and subtilisin family serine proteases,16,17 whereas
papain homologs do not show an elaborate hydrogen bond
network between the enzyme and the substrate backbone.10,18 Though placed on a very different scaffold, the
dominant S 1 specificity and b-sheet like backbone
hydrogen-bond interactions with the substrate demonstrates that the overall mode of substrate binding observed
with caspases has more in common with serine proteases
than other cysteine proteases. Thus, the caspases are very
distinct from the papain family proteases that display a
predominant P2 specificity and no b-sheet formation with
the substrate. The caspases can be considered as
proteases that have adopted a cysteine nucleophile to
hydrolyze proteins bound in a more-or-less serine protease
like substrate conformation.
The differences in the specificity of the caspases are
largely due to interactions at the distant S4 sub-site, which
results in the segregation of the caspases into three
different groups.19 Although this classification provides a
useful guide to specificity of the caspases it should be used
with caution, mainly because of the different level of activity
observed for the caspases. For example, the rate of
hydrolysis (kcat/KM) observed for caspase 1 on WEHDAMC is around 100-fold higher than that observed with
caspase-4 on the same substrate, 8 and thus, may
approach the rate by which caspases belonging to other
groups cleave the same substrate. Additionally, some
caspases, such as caspase 2, requires subsite occupancy
beyond S4 for efficient hydrolysis and thus their activity may
be misrepresented by the P4 categorization.20 While the
large degree of importance of the P4 interactions are well
described and clearly of biological importance, the
caspases are far from unique in their requirements. Indeed
many other proteases have a significant degree of
substrate discrimination at the P4 position. A prime
example of this is the subtilisin family which, though often
considered to have a rather broad primary (P1) specificity,
has a significant ability to discriminate between different P4
substituents.21
Proteases depend on transition state substrate binding
for catalysis, close to the tetrahedral intermediate, rather
than to the ground state of the native substrate. Thus, the
binding energy contained in the system is used to promote
catalysis rather than association with the substrate. This
means that proteases generally bind quite weakly to their
substrates in the ground-state (KM values in the mM range)
but very tightly to the transition state, which has important
implications for the generation of inhibitors of these
enzymes. Thus, in contrast to enzymes like kinases, the
generation of mutant substrates should not result in an
inhibitory form of the substrate, but merely in the particular
protein not showing any affinity for the protease any more.
Thus, the use of catalytically incapacitated mutants of
proteases as dominant negatives must be considered
limited due to the relative weak binding of substrates to
the ground state. Only caspases that are recruited, by virtue
of their N-peptides, to activation complexes should act as
dominant negative inhibitors of the activation process.
Enzymatic characteristics of the caspases
The features described above determine the enzymatic
properties of the caspases and thus, to a large extent their
biological function. As with other cysteine proteases the
caspases require a reducing environment in order to retain full
activity, presumably because the catalytic thiol is susceptible
to oxidation. This would tend to limit caspase activity to the
reducing environment found inside cells, and argue against
Catalytic properties of the caspases
HR Stennicke and GS Salvesen
1057
an extracellular role. It is clear from work on the recombinant
caspases that they require quite high concentrations of
reducing agents such as DTT to achieve maximal activity
(probably due to oxidation of the catalytic Cys during
purification; HR Stennicke and GS Salvesen, unpublished
results). The endogenous caspases in cell cytosols appear to
be fully active in the presence of low concentration of reducing
agents and recombinant material also retains full activity at
these conditions once activated providing that there are metal
chelators such as EDTA present (HR Stennicke and GS
Salvesen, unpublished results). This makes apparent a
related aspect to the oxidation, which is the effect of transition
metals on the activity. The influence of such metal ions on the
activity of other cysteine proteases has been well established
for a long time, predominantly due to the direct interaction with
the catalytic thiol but also the ability of these metals to
catalyze oxidation. It is therefore not surprising that the
caspases are sensitive to Zn2+, being completely inhibited in
mM range,7 although there are significant differences in their
affinity. Caspase 6 is most readily inhibited by Zn2+, becoming
completely inactivated by 0.1 mM, and caspase 3 is the least
sensitive requiring more than 1 mM for complete inactivation
in the presence of 20 mM 2-mercapto ethanol. The inhibition
of caspases by Zn2+ may explain the inhibitory action of this
metal on apoptosis,22,23 though the interaction is blocked by
thiol compounds,7 and therefore presumably highly dependent on the redox potential of the cell. Ca2+ has little effect on
the activity of the caspases at concentrations up to 100 mM,
although minor effect on the activity of caspase-7 have been
reported.8 Thus, the reported role of Ca2+ in apoptosis, see for
example,24 is unlikely to be due to any effect on the caspases.
Activation of the caspases
In general the activation of most proteases takes place by
limited proteolysis leading to removal of an N-terminal
activation peptide, also known as the propeptide. This
mechanism of activation is used by the majority of all known
proteases with the notable exception of the caspases. In the
papain like enzymes such as cathepsin B and L the
propeptide binds in the active site cleft much like a substrate
but in reverse direction, physically preventing access to the
substrate binding site.25,26 In this case the activation occurs
by auto-catalytic removal of the propeptide in trans (an
intermolecular reaction). In contrast the propeptides of the
serine proteases a-lytic protease and subtilisin bind more like
a natural substrate and the activation is unregulated,
occurring by cleavage of the bond between the residues
occupying the S1 and S1' subsites of the enzymes.27 ± 30 This
reaction occurs in cis (an intra molecular reaction). Interestingly the propeptides of all these proteases are potent
inhibitors of the mature enzymes and usually characterized
by low to sub-nano molar binding constants.31 ± 34
In the activation mechanism utilized by members of the
chymotrypsin family the propeptide does not physically block
the active site. Here activation occurs when propeptide
cleavage reveals a new N-terminal a-amino group that inserts
into the core of the protein, and secures formation of the
active site.35 ± 37 In cases where such events are required it
has been demonstrated that addition of specific peptides can
activate the proteases, the perhaps most well known
example being that of trypsin.38 Recently, an analogous
mode of activation has been suggested for the caspases in
the activation of caspase-3 by RGD containing peptides.39
The mechanism of this activation, which is postulated to be a
direct effect, is unclear and indeed attempts in our laboratory
to repeat the original observation using cytosolic extracts as
well as immuno-precipitated or purified recombinant procaspase-3 have failed (HR Stennicke, QL Deveraux and GS
Salvesen, unpublished results).
In contrast to other mechanisms, caspase activation
primarily occurs through cleavage within a segment at an
internal position in the zymogen. In caspase 1, removal of the
N-peptide has been implicated as a maturation step,40 but
simply removing the N-peptide is not sufficient for activation.
On the other hand, the biochemical properties of the short Npeptide caspases such as caspase-3, which are involved in
the execution of the apoptotic signal, are not affected by the
removal of the N-peptide.41 Additionally, caspase-9, which is
a key initiator of the post-mitochondrial cell death pathway,
does not get its large N-peptide removed during activation.42 ± 45 Thus the function of the individual N-peptide
extensions of the caspases is not conserved.
It is generally agreed that initiator caspases transmit the
proteolytic signal by directly processing executioner
zymogens, and at least in vitro activation of the
executioner caspase zymogens by the initiators is efficient
and fast (Table 1). However, the molecular mechanism
leading to the active enzyme is currently not known since
there are little available biochemical data and no structural
models available for the zymogens. One possibility is that
the cleavage of the inter-domain linker results in formation
of the catalytic dyad, which is distorted in the zymogen due
to tension in the protein. The degree of distortion imposed
on the catalytic dyad would then be a function of the length
of the inter-domain linker and indeed caspases containing
relatively long inter-domain linkers (caspases 8 and 9) have
been demonstrated to possess activity in their zymogen
forms. Possibly this is a key to the mechanism by which
activation occurs in the activator complexes.
The importance of zymogenicity
Interestingly, depending upon expression conditions of
recombinant proteins, one can obtain either processed active
caspase or unprocessed zymogen from the same construct,
at least for caspases 3, 7 and 9.41,45,46 For example, a short
induction time (530 min) yields unprocessed zymogens but
longer ones (43 h) yield fully processed enzymes. SignifiTable 1 Activation rates of executioner caspase zymogens by selected initiators
(M71 s71)
Pro-caspase 3
Pro-caspase 7
Caspase 8
Caspase 10
Granzyme B
2.26106
4.46106
0.76106
1.26106
4.86106
6.46106
Puri®ed recombinant zymogens of the executioner caspases 3 and 7 are
activated most ef®ciently by the serine protease granzyme B, followed by the
apical caspase 8, and then caspase 10. Data for caspase-3 are taken from41
and for caspase-7 from HR Stennicke and GS Salvesen, unpublished results
Catalytic properties of the caspases
HR Stennicke and GS Salvesen
1058
cantly, even very short expression times and low inducer
concentrations have failed to yield caspase 8 zymogens in our
hands (HR Stennicke and GS Salvesen, unpublished data).
Caspase 8 processes itself extremely rapidly upon heterologous expression in E. coli, suggesting that the zymogen
must possess significant intrinsic proteolytic activity, allowing
for autoprocessing. Indeed, a non-processing caspase 8
zymogen possesses 1% of the activity of the fully processed
enzyme,47 a very substantial activity compared with most
protease zymogens. These observations are the basis for the
induced proximity hypothesis for the in vivo activation of
caspase 8, whose assembly is forced by ligation of death
receptors in a process mediated by specific adaptor
proteins.48 This clustering of zymogens possessing intrinsic
enzymatic activity forces processing in trans, and activation of
the first protease in the death receptor pathway to
apoptosis.47,49,50 An analogous activation of pro-caspase 9
by zymogen clustering may account for the origin of the postmitochondrial death pathway,51,52 and the mechanism may
be highly conserved since the worm caspase CED-3 may be
similarly activated.53,54 These observations stress the
importance of zymogenicity (defined in Table 2) in triggering
the cell death signaling pathway.
The interesting range of zymogenicity values displayed
by members of the caspase family is mirrored by members
of the chymotrypsin family of serine proteases, with trypsin
and tissue plasminogen activator (tPA) shown for comparison (Table 2). Presumably enzymes such as tPA and
caspase 9 have dispensed with the requirement for
proteolysis as a mechanism of substantially increasing
their activities, because allosteric regulators substitute this
function ± fibrin for tPA and Apaf-1 for caspase 9. In the
case of tPA, specific side-chain interactions, absent in other
members of the chymotrypsin family, allow activity of the
zymogen. However, in the absence of a molecular structure
of the caspase 8 and 9 zymogens, little evidence is
available to explain the high activity of the unprocessed
protein. One clue is suggested by the structure of active
caspases 1 and 3, each composed of two catalytic units
thought to arise from dimerization of monomeric zymogens
(reviewed in55). If activation of zymogens of the initiator
caspases 8, 9 and CED-3 operates by clustering, then the
clustering phenomenon may be explained by adapterdriven homo-dimerization of monomers.
Origin of the caspases?
A puzzling feature in the caspases is the current absence of
any reported homologs outside of this tightly knit family
Table 2 Zymogenicity of selected proteases
Zymogenicity
Caspase Caspase Caspase
3
8
9
Trypsin
tpA
410,000
410,000
2 ± 10
100
10
Zymogenicity is the ratio of the activity of a processed protease to the activity of
the zymogen on any given substrate. It is a measure of how effectively the
zymogen is constrained, with a large number corresponding to insigni®cant
activity of the zymogen. Data for trypsin and tissue plasminogen activator (tPA)
are taken from58
restricted to animals. There should be homologs consisting of
the same fold that would help to shed light on the origin of the
family. One of the most notable properties of the caspases is
the dominance of the S1 pocket, which caspases share in
principle with serine proteases of the chymotrypsin family, and
in particular with granzyme B.56 Does this principle extend to
other cysteine protease families that also have a dominant P1
specificity? Are there other families that use a dominant S1
pocket, but with different specificity, positioned on the
currently unique caspase fold?
Such a possibility has been proposed for the bacterial
proteases clostripain and R-gingipain (dominant P 1
specificity for Arg), K-gingipain (dominant P1 specificity for
Lys), and the plant and animal legumains (dominant P1
specificity for Asn). Though all are cysteine proteases, they
may share the caspase fold, and therefore represent very
distant homologs.57 Thus, we may so far only have found a
few of the members of the family sharing the caspase fold
and may still find a number of proteases that will eventually
close the evolutionary gap between the bacterial and the
mammalian members.
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