(Received for publication, August 23, 1996, and in revised form, November 4, 1996)
From the Department of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Human chymase and rat chymase-1 are mast cell
serine proteases involved in angiotensin II (Ang II) formation and
degradation, respectively. Previous studies indicate that both these
enzymes have similar P1 and P2 preferences,
which are the major determinants of specificity. Surprisingly, despite
the occurrence of optimal P2 and P1 residues at
the Phe8 and Tyr4
bonds
(where
, indicates the scissile bond in peptide
substrates) in Ang I (DRVYIHPFHL), human chymase cleaves the
Phe8
bond with an ~750-fold higher catalytic
efficiency (kcat/Km) than
the Tyr4
bond in Ang II (DRVYIHPF), whereas rat
chymase-1 cleaves the Tyr4
bond with an ~20-fold
higher catalytic efficiency than the Phe8
bond.
Differences in the acyl groups IHPF and DRVY at the
Phe8
and Tyr4
bonds,
respectively, are chiefly responsible for the preference of human
chymase for the Phe8
bond. We show that the IHPF
sequence forms an optimal acyl group, primarily through synergistic
interactions between neighboring acyl group residues. In contrast to
human chymase, rat chymase-1 shows a preference for the
Tyr4
bond, mainly because of a catalytically
productive interaction between the enzyme and the P
1
Ile5. The overall effect of this P
1 Ile
interaction on catalytic efficiency, however, is influenced by the
structure of the acyl group and that of the other leaving group
residues. For human chymase, the P
1 Ile interaction is not
productive. Thus, specificity for Ang II formation versus
Ang II degradation by these chymases is produced through synergistic
interactions between acyl or leaving group residues as well as between
the acyl and leaving groups. These observations indicate that
nonadditive interactions between the extended substrate binding site of
human chymase or rat chymase-1 and the substrate are best explained if
the entire binding site is taken as an entity rather than as a
collection of distinct subsites.
Chymases1 are a family of mast cell
serine proteinases involved in such diverse functions as inflammation
(1), parasite expulsion (2), and peptide hormone processing (3-5).
These serine proteinases are synthesized as inactive precursors but are
stored in secretory granules as active enzymes (6). Recent phylogenetic
evidence indicates that mammalian chymases occur as two distinct
isoenzyme groups, and
(7).
-Chymases include human chymase,
dog chymase, mouse chymase-5, rat chymase-3, and gerbil chymase-2 (7,
8).
-Chymases include rat chymase-1 and -2, mouse chymase-1, -2, -4, and -L, and gerbil chymase-1 (7, 8). Kinetic studies indicate that
-
and
-chymases differ in their substrate specificity. For example,
human chymase efficiently converts the decapeptide Ang
I2 to the octapeptide hormone Ang II by
splitting the Phe8-His9 bond in Ang I (5). The
generated Ang II is not further degraded because the
Tyr4-Ile5 bond in Ang II is resistant to
cleavage by human chymase. Histochemical (9) and in vivo
functional studies (10) suggest a major role for primate chymases in
regulating tissue Ang II levels. Rat chymase-1, in contrast, is an
angiotensinase because it readily splits the Tyr4-Ile5 and the
Phe8-His9 bonds in angiotensins (3, 7).
Early comparative studies on chymase specificities by Powers et
al. (11) using peptide 4-nitroanilide substrates indicated that
the S1 to S4
subsites3 of human chymase and rat
chymase-1 are similar. In both human chymase and rat chymase-1, the key
features for optimal acyl group interactions are a P1
hydrophobic aromatic residue, a P2 hydrophobic residue or
Pro, and a P3 hydrophobic residue. S4 subsite
interactions are less restrictive. Thus, these studies could not
explain why human chymase is an Ang II-forming enzyme and rat chymase-1
is an angiotensinase (3, 5) and suggested to us that enzyme-substrate interactions other than those occurring at the S1 to
S4 subsites of these enzymes could be important for
determining specificity. In a previous paper we explored the
S1 subsite as well as the S1 to
S
2 subsites of human chymase using decapeptide Ang I
analogs. We showed that a P1 hydrophobic aromatic residue
was necessary but that several nonconservative changes in
P
1 and P
2 positioned residues produced small
effects (i.e. a ~3-fold change in the specificity constant
kcat/Km) on the cleavage of
the Phe8
Xaa9 bond (12). To determine
if the Tyr-Ile P1-P
1 combination forms a poor
cleavage site for human chymase or if the context of the bond within
the substrate is important, we synthesized an peptide analog of Ang I
that contained two Tyr-Ile bonds, one that naturally occurs at the 4-5
position and one introduced at the 8-9 position. The
Tyr4-Ile5 bond was resistant to cleavage by
human chymase, but the Tyr8-Ile9 bond was
readily cleaved; the difference in
kcat/Km for the cleavage of
the Tyr8-Ile9 bond versus the
Tyr4-Ile5 bond was >500:1 (12). We proposed,
therefore, that for human chymase, the structural context of the
scissile bond within the polypeptide substrate is likely to be an
important determinant of specificity. What is the structural context
that makes the Phe8-His9 bond in Ang I,
relative to the Tyr4-Ile5 bond in Ang II,
highly susceptible to cleavage by human chymase? We show that in human
chymase this structural context is generated through synergistic
interactions between neighboring P4 to P1 residues of the substrate acyl group. Rat chymase-1, on the other hand,
has a distinct P
1 preference that distinguishes its
specificity from human chymase.
Peptides used in this study were synthesized by The Protein Core Facility of The Cleveland Clinic Foundation. Peptides were purified (purity >99%) on a C18 reverse phase HPLC column and characterized by amino acid analysis and by analytical C18 reverse phase HPLC. Peptide concentrations were standardized by amino acid analysis.
Enzymes and Enzyme KineticsHuman chymase was purified to
homogeneity from human left ventricular tissue (5). Rat chymase-1 was
purified from rat peritoneal mast cells as described previously by Le
Trong et al. (3). Identity of these enzymes was established
by N-terminal sequence analysis. To determine Km and
Vmax values for the human chymase and rat
chymase-1 reactions, initial velocities (v) were determined
as described by us previously (12). Fifteen concentrations of substrate
ranging between 0.8 and 1,000 µM with human chymase or
rat chymase-1 were incubated at 37 °C in 20 mM Tris-HCl
buffer, pH 8.0, containing 0.5 M KCl, and 0.01% Triton
X-100 (final volume, 50 µl) for 20 min (in the cleavage of Ang II by
human chymase a 120-min incubation period was used). For each peptide
substrate, enzyme concentration was adjusted to between 0.02 and 4 nM to ensure that <15% of the substrate was utilized at
the lowest substrate concentration. Under these conditions, product
formation was linear with respect to time over the duration of the
incubation. Reactions were terminated by the addition of 300 µl of
ice-cold ethanol, and the resulting solution was evaporated to dryness.
The residue was resuspended in 125 µl of distilled water, and 100 µl was applied to a C18 reverse phase HPLC column (Vydac,
Hesperia, CA). The column was developed with linear acetonitrile
gradients containing either 25 mM triethylammonium
phosphate buffer, pH 3.0, 0.1% trifluoroacetic acid, or 5 mM hexane sulfonic acid at a flow rate of 2 ml/min. The
column effluent was monitored at 214 nm. The elution positions of Ang I
and of Ang II and its analogs were also determined using pure synthetic
standards. The peak area corresponding to Ang II or its analog was
integrated to calculate Ang II or Ang II analog formation. Products
were separated by reverse phase HPLC and identified by amino acid
analysis. Km and Vmax values
were calculated by nonlinear regression using the equation
v = Vmax × [S]/(Km + [S]). Correlation coefficients were
routinely >0.99 but never <0.97. The concentration of human chymase
and rat chymase-1 was determined using bicinchoninic acid protein assay
reagent (Pierce) with pure bovine -chymotrypsin as the standard. The
overall rate constant kcat was calculated by the
formula kcat = Vmax/[E0], where
[E0] is the total enzyme concentration.
GT
,
Gbinding, and
Gcat were calculated using the equations
GT
= [
RT ln
(kcat/Km)substituent peptide]
[
RT ln
(kcat/Km)parent peptide],
Gbinding = [
RT ln
(1/Km)substituent peptide]
[
RT ln
(1/Km)parent peptide], and
Gcat = [
RT ln
(kcat)substituent peptide]
[
RT ln
(kcat)parent peptide],
respectively, as described by Wells (13).
Gbinding and
Gcat represent the difference between two
substrates in the free energy required to form the enzyme-substrate complex (ES) from E + S and to convert the ES complex to the
ES
complex, respectively.
Human chymase
converts Ang I to Ang II by splitting the Ang I
Phe8-His9 bond with high catalytic efficiency
(kcat/Km = 3.6 µM1·s
1). At a human chymase
concentration (~20 pM) that allows a rapid and efficient
cleavage of this bond in Ang I, the chymotrypsin-sensitive bond in Ang
II (i.e. the Tyr4-Ile5 bond) is not
appreciably cleaved. Using a high concentration of human chymase (~4
nM) and prolonged incubation times, we determined that the
catalytic efficiency and the overall rate constant
kcat for the cleavage of the Ang II
Tyr4-Ile5 bond are ~780-fold and
~3,500-fold lower, respectively, than that for the cleavage of the
Ang I Phe8-His9 bond (Table I).
Because previous studies indicated that the P1,
P2, and P
1 residues at both the
Try4 and Phe8 bonds are near optimal for human
chymase (11, 12), we considered whether the acyl group and/or the
leaving group at these cleavage sites as a whole could explain these
remarkable differences in kcat and catalytic
efficiency.
|
To determine if the side chain interactions provided by the Ang I
leaving group His-Leu influence catalysis, we compared the cleavage by
human chymase of DRVYIHPFHL (Ang I) with that of
DRVYIHPF
GG (peptide hc1). Table I shows that these
peptides are cleaved with similar catalytic efficiency, indicating that
His-Leu side chain interactions are not important for catalysis. The
effect of backbone leaving group interactions on catalytic efficiency is summarized in Table I. Fewer than two backbone interactions produce
a decrease in catalytic efficiency, e.g. cleavage of the Phe8-Gly9 bond in
DRVYIHPF
G (peptide hc2, also see hc3) occurs with
a ~55-fold lower catalytic efficiency than of the
Phe8-Gly9 bond in
DRVYIHPF
GG (peptide hc1), and an increase in
leaving group backbone interactions beyond those provided by two
residues was without effect (peptides hc4, hc5, and hc6). Thus,
backbone interactions provided by the His-Leu leaving group are
necessary for the efficient catalysis of the
Phe8-His9 bond but side chain interactions are
not important.
Ang II degradation by human chymase (DRVYIHPF;
kcat/Km = 0.0046 µM
1·s
1) occurs with a
~780-fold lower catalytic efficiency than Ang II formation. In Ang II
degradation, IHPF forms the leaving group. Cleavage of
DRVY
GG (peptide hc7), an Ang II analog in which
the leaving group side chains and the nonessential part of the peptide
backbone are deleted, occurs with an ~11-fold higher catalytic
efficiency than the cleavage of Ang II (Table I). Effect of individual
leaving group side chain interactions on Ang II catalysis by human
chymase are summarized in Table I. Deletion of P
1 (peptide
hc8), P
2 (peptide hc9), P
3 (peptide hc10), or
P
4 (peptide hc11) side chains in Ang II leads to a
2-14-fold increase in Km, a 7-170-fold increase in
kcat, and a 2-15-fold increase in
kcat/Km. One way to interpret
these data4 is that the binding energy
imparted by these P
interactions leads to a more stable ES complex,
but the stability of the ES
complex is decreased. The
activation energy of the kcat therefore increases substantially, so the reaction rate and catalytic efficiency both decrease. Thus, the IHPF leaving group at the Ang II
Tyr4
bond is detrimental for catalysis, and all
leaving group side chains contribute to this effect.
In Ang II formation, the sequence DRVYIHPF (Ang II) forms the acyl
group. Table I shows that catalytic efficiency does not change when
this Ang I acyl group is reduced in length from DRVYIHPF to IHPF
(peptide hc12). IHPFHL (peptide hc12) and
IHPF
GG (peptide hc13) are cleaved with similar
catalytic efficiencies (Table I), again indicating that side chain
interactions of the His-Leu leaving group are not consequential in the
cleavage of the Phe8-His9 bond. Therefore, of
the acyl group interactions, those provided by the Ang I residues IHPF
are sufficient for the high catalytic efficiency with which human
chymase cleaves the Phe8-His9 bond in Ang I. Because the acyl group involved in Ang II degradation, i.e.
DRVY, is also four residues long, in the next series of experiments we
directly compared the influence of the IHPF and DRVY acyl groups on
catalytic efficiency.
The catalytic efficiency for human chymase-dependent
cleavage of IHPFGG (peptide hc13) was ~45-fold
higher than for DRVY
GG (peptide hc7) cleavage
(Table I). This difference in catalytic efficiency was almost entirely
due to an ~44-fold difference in kcat,
indicating that the IHPF acyl group, relative to the DRVY acyl group,
does not affect the stability of the ES, but instead its binding energy
is realized only in the ES
, i.e. the
structure of the acyl group binding site is much more complementary to
the transition state structure of the IHPF acyl group than to that of
the DRVY acyl group.
The difference in DRVY and IHPF acyl groups reactivities was surprising
because in both these acyl groups the P1 and P2
residues were previously predicted to be optimal (11). This observation prompted us to examine which residue(s) was the chief determinant of
the reactivity difference observed between these acyl groups. Table
II shows that replacement of the P4 Asp with
Ile (peptide hc15), P3 Arg with His (peptide hc16), or
P2 Val with Pro (peptide hc17) in the DRVY acyl group of
Ang II produced decreases in the free energy required to reach the
ES during hydrolysis
(
GT
=
6.37
kJ·mol
1 for P4 Asp
Ile,
2.01
kJ·mol
1 for P3 Arg
His, and
0.67
kJ·mol
1 for P2 Val
Pro). Replacement of
the P1 Tyr with Phe (peptide hc18) in the DRVY acyl group
of Ang II produced a small increase (0.71 kJ·mol
1) in
GT
. Therefore, with respect
to individual component differences between the DRVY and IHPF acyl
groups, the P4 Asp
Ile change has the most favorable
effect on reactivity. Additivity analysis indicates a good agreement
between the observed [
GT
(multiple)] and calculated
[
GT
(components)]
decreases in transition state stabilization energy for two- or
three-component transitions in the Ang II acyl group, e.g.
DRVY
IHVY, DRVY
DRPF, or DRVY
IHVF (peptides
hc19-21) (Table II). Remarkably, however, when we compared the
observed decrease in transition state stabilization energy associated
with the DRVY
IHPF change in the Ang II acyl group
[
GT
(multiple) =
14.3
kJ·mol
1] with that calculated on the basis of
individual P4 to P1 changes [
GT
(components) =
8.34 kJ·mol
1], it is apparent that a
significant component (>40%) of the high reactivity of the IHPF
acyl group for human chymase is due to synergistic behavior between all
four these acyl group residues. Additional additivity analyses based on
calculations of
Gbinding (from
Km values in Table I) and
Gcat (from kcat
values in Table I) indicates that for the DRVY
IHPF
IHPF
IHPF transition there is good
agreement between observed and calculated measurements of
Gbinding
[
Gbinding (multiple)
Gbinding (components) = 0.95 kJ·mol
1], but deviations from simple additivity are
clearly evident in
Gcat measurements
[
Gcat (multiple)
Gcat (components) =
6.97
kJ·mol
1]. Thus, synergistic behavior of the IHPF acyl
group residues in producing a highly reactive human chymase substrate
is seen in the energetics to reach ES
complex but is not
observed in the initial energetics of substrate binding that leads to
the formation of the ES complex.
|
We speculate that the extended substrate binding site of human chymase,
particularly the region that binds the P4 to P1
acyl group residues, has specialized to allow the
Phe8His9 bond in Ang I to bind in a
highly productive mode. This optimal acyl group is generated through
synergistic interactions between neighboring acyl group residues; these
interactions, we believe, form the basis of the "structural
context" that has allowed human chymase to become an efficient Ang
II-forming enzyme. The critical nature of these synergistic
interactions in determining specificity is illustrated by the fact that
the Ang II Tyr4-Ile5 bond and the Ang I
Phe8-His9 bond, which seemingly contain optimal
P1 and P2 residues and near optimal
P
1 residues (11, 12), are cleaved by human chymase with an
~3,500-fold difference in kcat and an
~780-fold difference in
kcat/Km.
In rat
chymase-1-dependent Ang I degradation, the product DRVY
accumulates ~20-fold faster than Ang II (7), suggesting that rat
chymase-1 splits the Ang I Tyr4-Ile5 bond
~20-fold faster than the Phe8-His9 bond.
Table III shows that rat chymase-1 cleaves the
Tyr4-Ile5 bond in Ang II with a ~20-fold
higher catalytic efficiency than the Phe8-His9
bond in IHPFHL (peptide rc1). Therefore, we used
DRVY
IHPF and IHPF
HL as model
peptides to study the essential components of acyl and leaving group
interactions that are involved in the cleavage of Ang I
Tyr4-Ile5 and Phe8-His9
bonds.
|
No difference in the catalytic efficiency was observed between
IHPFHL (peptide rc1) and
IHPF
GG (peptide rc2) cleavage by rat chymase-1
(Table III). Catalytic efficiency was also not affected when backbone
leaving group interactions were increased from those provided by two
residues to those provided by five residues; an example of this using
the IHPF acyl group is shown in Table III (peptides rc2, rc3, rc4, and
rc5). DRVY
GG (peptide rc6) cleavage by rat
chymase-1 occurs with a ~22-fold lower catalytic efficiency than
DRVY
IHPF cleavage. This decrease in catalytic
efficiency is due to a 3.2-fold increase in Km and
an 6.7-fold decrease in kcat. These results
indicate that leaving group side chain interactions overall facilitate
rat chymase-1-dependent catalysis of the
Tyr4-Ile5 bond in Ang II but not the
Phe8-His9 bond in Ang I.
Table III summarizes the effect of leaving group side chain
interactions on Ang II catalysis by rat chymase-1. Deletion of the
P1 Ile side chain in Ang II (peptide rc7) produced a
6.5-fold decrease in catalytic efficiency, but deletion of the
P
2 His (peptide rc8), P
3 Pro (peptide rc9),
or P
4 Phe (peptide rc10) side chains produced a
3.8-6.6-fold increase in catalytic efficiency. These individual
P
1 to P
4 side chain deletions produced small (<2.2-fold) effects on Km. Additivity analysis
predicts that the cumulative detrimental effects of the Ang II
P
2-, P
3-, and P
4-side chains on
transition state stabilization energy
[
GT
(P
2,
P
3, P
4) = 12.28 kJ·mol
1]
should overcome the decrease in transition state stabilization energy
produced by the P
1 Ile side chain
[
GT
(P
1) =
4.84 kJ·mol
1] to generate an ~7.5
kJ·mol
1 increase in the free energy required to reach
the ES
complex during hydrolysis (Table
IV). However, the overall observed effect of Ang II
leaving group interactions on catalytic efficiency is favorable
(
GT
=
7.94
kJ·mol
1) (Table IV); this is evident when Ang II
cleavage (kcat/Km = 0.085 µM
1·s
1) is compared to
DRVY
GG cleavage (peptide rc6;
kcat/Km = 0.0039 µM
1·s
1). These findings
suggest that interaction of the Ang II leaving group with the rat
chymase-1 S
subsite is dependent on secondary structure of the
substrate leaving group or perhaps that of the entire substrate and
that the P
1 interaction is dominant over other P
interactions. These speculations led us 1) to consider whether the
effect of the Ang II leaving group would be different if this leaving
group was attached to a different acyl group and 2) to examine the
effect of a single P
1 Ile side chain on catalysis.
|
Direct comparisons were made between the Ang II acyl group DRVY and the
Ang I acyl group IHPF. Specificity constants for the cleavage of
IHPFGG (peptide rc2) and
DRVY
GG (peptide rc6) were identical (Table III),
indicating that within the context the Gly-Gly leaving group rat
chymase-1 does not differentiate between these acyl groups. To
determine if interactions between the acyl and the leaving group can
influence transition state stabilization, we compared the effects of
IHPF and Gly-Gly leaving groups on DRVY and IHPF acyl groups (peptides
rc2, rc6, rc11, and Ang II). Table IV shows that the IHPF leaving
group, relative to the Gly-Gly leaving group, causes a
7.94
kJ·mol
1 decrease in
GT
when DRVY is the acyl
group and a
13.8 kJ·mol
1 decrease in
GT
when IHPF is the acyl
group. Thus, acyl-leaving group interactions can greatly influence the
overall effect of the leaving group on transition state
stabilization.
To show if the favorable effect of the IHPF leaving group in
IHPFIHPF catalysis can be mimicked by the
introduction of an Ile side chain at the P
1 position, we
examined IHPF
IHPF (peptide rc11),
IHPF
GGGG (peptide rc4),
IHPF
IGGGG (peptide rc12), and
IHPF
GGGGG (peptide rc5) catalysis by rat
chymase-1. IHPF
IHPF was cleaved by rat chymase-1
with a 138-fold higher catalytic efficiency than
IHPF
GGGG. IHPF
IGGGG was
cleaved with a 66-fold higher catalytic efficiency than IHPF
GGGGG; peptides in which Ile side chains were
introduced at additional leaving group sites, e.g.
IHPF
IGIGG and IHPF
IGIGI
(peptides rc13 and rc14), were catalyzed with an efficiency similar to
that of IHPF
IGGGG (Table III). These findings are
consistent with the view that the P
1 Ile side chain of the
IHPF leaving group provides the dominant favorable effect. This Ile
effect on catalytic efficiency is produced by a 12.5-fold decrease in
Km and a 5.3-fold increase in
kcat (peptides rc5 and rc12) and is
position-dependent. In contrast, in the case of human
chymase, introduction of an Ile in the P
1 position of the
pentaglycyl leaving group (peptides hc6 and hc22) generates a
nonproductive binding mode, because this interaction produces an
~3-fold decrease in Km, but
kcat/Km decreases by a small
extent (~2.7-fold) (Table I).
In these studies with rat chymase-1, we show that the introduction of a
single P1 Ile side chain on a pentaglycyl leaving group
markedly increases catalytic efficiency when IHPF is the acyl group;
also, we show using the DRVY acyl group that deletion of the
P
1 Ile side chain in the IHPF leaving group decreases catalytic efficiency. We believe that the active site of rat chymase-1 does not specifically favor the
-branched aliphatic side chain of
P
1 Ile but that hydrophobic side chains are generally
preferred here. In this regard, Le Trong et al. (3) have
reported that rat chymase-1-dependent cleavage of peptides
and proteins is more likely to occur between pairs of hydrophobic
residues, where the P
1 residue is Ile, Leu, Phe, Trp, or
Tyr. Because interactions between acyl and leaving groups and between
leaving group residues can influence substrate catalysis by rat
chymase-1, the contribution of this P
1 interaction to
overall catalytic efficiency is likely to depend on the structure of
the rest of the substrate. Despite its slight preference for the IHPF
acyl group over the DRVY acyl group, it is likely that rat chymase-1
shows an ~20-fold preference for the
Tyr4-Ile5 bond over the
Phe8-His9 bond because a P
1 Ile
exists at the Tyr4-Ile5 bond of Ang I.
Early studies on chymotrypsin and
trypsin show that the main feature of serine protease specificity in
these digestive enzymes is the interaction of the P1
substrate residue with the S1 subsite on the enzyme (14,
15). The S1 subsite is a deep pocket whose structure has
been defined through crystallography and mutagenesis, and amino acid
side chains that fit best in this pocket have been defined by examining
the effect of substrate variations in the P1 position on
catalytic efficiency. More recent studies show that several regulatory
serine proteases have a S1 subsite preference similar to
that of chymotrypsin or trypsin but show considerable preference
between two or more optimal P1 residues in polypeptide substrates. It is now clear that trypsin and chymotrypsin can also
distinguish between two or more optimal P1 residues in
polypeptide substrates. To understand the basis for this preference,
additional S and S subsites have been examined for these enzymes using
a linear approach, where the influence of several side chains at each P
or P
position on catalysis is systematically analyzed; that is, in a
polypeptide consisting of n residues, n
1 residues are kept constant, and one residue is varied at a time.
Implicit in this widely used approach is the view that a distinct
Sx or S
x subsite exists for each
Px or P
x residue, respectively. This view is
supported by the finding that observed changes in transition state
stabilization energies due to multiple P or P
substitutions in a
substrate can be predicted from the sum of transition state stabilization energies calculated from individual changes,
i.e. the S and S
subsites function independently. For
example, simple additive behavior is seen with substrate hydrolysis by
trypsin (16), chymotrypsin (17), tissue-type plasminogen activator (16), and subtilisin (18). Nonadditive interactions, i.e. subsite interdependence in transition state stabilization, has also
been observed with subtilisin and chymotrypsin in the hydrolysis of
some substrates (18, 19). In most examples of subsite interdependence, however, it is apparent that the function of one residue is compromised by mutation of another (13, 18, 19). In contrast, using Ang II
formation versus Ang II degradation as examples, we show that human and rat chymase-1 specificities are achieved through synergistic interactions between neighboring residues of the acyl group
or the leaving group as well as interactions between these groups.
These observations indicate that interactions between the binding site
of human chymase or rat chymase-1 and the substrate are best explained
if the entire substrate binding site is taken as an entity rather than
as a collection of distinct Sx and S
x subsites. Thus, these studies suggest that the identification of highly
reactive novel substrates for human chymase as well as the design of
substrate-derived inhibitors cannot be predicted from simple subsite
mapping; on the other hand, combinatorial approaches are likely to be
effective. This speculation is strengthened by the recent studies of
Bastos et al. (20) that show highly synergistic behavior
between certain P3-P2 combinations in
combinatorial human chymase inhibitor libraries that could not be
predicted from simple subsite maps of human chymase (11). Combinatorial approaches could also prove be useful in defining the specificity of
related leukocyte serine proteases such as cathepsin G where linear
approaches have failed in identifying highly efficient natural
substrates.
We thank Drs. R. Ramchandran and C. B. Post for critical reading of our manuscript and Dr. K. S. Misono for peptide synthesis. The assistance of Christine Kassuba in manuscript editing is kindly acknowledged.