Distinct Multisite Synergistic Interactions Determine Substrate Specificities of Human Chymase and Rat Chymase-1 for Angiotensin II Formation and Degradation*

(Received for publication, August 23, 1996, and in revised form, November 4, 1996)

Subramaniam Sanker , Unnikrishnan M. Chandrasekharan , Dennis Wilk , Manuel J. Glynias , Sadashiva S. Karnik and Ahsan Husain Dagger

From the Department of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 Phe8down-arrow and Tyr4down-arrow bonds (where down-arrow , indicates the scissile bond in peptide substrates) in Ang I (DRVYIHPFHL), human chymase cleaves the Phe8down-arrow bond with an ~750-fold higher catalytic efficiency (kcat/Km) than the Tyr4down-arrow bond in Ang II (DRVYIHPF), whereas rat chymase-1 cleaves the Tyr4down-arrow bond with an ~20-fold higher catalytic efficiency than the Phe8down-arrow bond. Differences in the acyl groups IHPF and DRVY at the Phe8down-arrow and Tyr4down-arrow bonds, respectively, are chiefly responsible for the preference of human chymase for the Phe8down-arrow 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 Tyr4down-arrow 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.


INTRODUCTION

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, alpha  and beta  (7). alpha -Chymases include human chymase, dog chymase, mouse chymase-5, rat chymase-3, and gerbil chymase-2 (7, 8). beta -Chymases include rat chymase-1 and -2, mouse chymase-1, -2, -4, and -L, and gerbil chymase-1 (7, 8). Kinetic studies indicate that alpha - and beta -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 S'1 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 Phe8down-arrow 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.


EXPERIMENTAL PROCEDURES

Peptides

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 Kinetics

Human 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 alpha -chymotrypsin as the standard. The overall rate constant kcat was calculated by the formula kcat = Vmax/[E0], where [E0] is the total enzyme concentration. Delta Delta GTDagger , Delta Delta Gbinding, and Delta Delta Gcat were calculated using the equations Delta Delta GTDagger  = [-RT ln (kcat/Km)substituent peptide- [-RT ln (kcat/Km)parent peptide], Delta Delta Gbinding = [-RT ln (1/Km)substituent peptide- [-RT ln (1/Km)parent peptide], and Delta Delta Gcat = [-RT ln (kcat)substituent peptide- [-RT ln (kcat)parent peptide], respectively, as described by Wells (13). Delta Delta Gbinding and Delta Delta 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 ESDagger complex, respectively.


RESULTS AND DISCUSSION

Why Human Chymase Is an Ang II-forming Enzyme

Human chymase converts Ang I to Ang II by splitting the Ang I Phe8-His9 bond with high catalytic efficiency (kcat/Km = 3.6 µM-1·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.

Table I.

Hydrolysis of Ang I, Ang II, and their analogues by human heart chymase

The kinetic constants were determined in 20 mM Tris-HCl buffer, pH 8.0, containing 0.5 M KCl and 0.01% Triton X-100 at 37 °C. Km and kcat values were determined by nonlinear regression; values are the means ± S.D. of three independent determinations for each peptide.
Peptide name/ reference number Substrate (Pn, P3, P2, P1down-arrow P'1, P'2, P'3, P'n)a Km kcat kcat/Km

µm s-1 µm-1·s-1
Ang I DRVYIHPFdown-arrow HL 40  ± 2.2 146  ± 3.3 3.6
Ang II     DRVYdown-arrow IHPF 8.9  ± 2 0.041  ± 0.003 0.0046
hc1 DRVYIHPFdown-arrow GG 49  ± 0.25 330  ± 0.4 6.7
hc2 DRVYIHPFdown-arrow G 240  ± 2.5 29  ± 0.1 0.12
hc3 DRVYIHPFdown-arrow G-NH2 99  ± 3.3 100  ± 1.4 1.0
hc4 DRVYIHPFdown-arrow GGG 48  ± 0.5 440  ± 5.0 9.0
hc5 DRVYIHPFdown-arrow GGGG 62  ± 0.5 350  ± 6.4 5.6
hc6 DRVYIHPFdown-arrow GGGGG 54  ± 2.0 420  ± 6.6 7.7
hc7     DRVYdown-arrow GG 125  ± 7.6 6.4  ± 0.06 0.051
hc8     DRVYdown-arrow GHPF 31  ± 1.4 0.29  ± 0.015 0.0093
hc9     DRVYdown-arrow IGPF 56  ± 0.45 2.5  ± 0.15 0.045
hc10     DRVYdown-arrow IHGF 20  ± 0.46 0.56  ± 0.04 0.028
hc11     DRVYdown-arrow IHPG 102  ± 11 7.0  ± 0.5 0.068
hc12     IHPFdown-arrow HL 78  ± 2.3 274  ± 5 3.5
hc13     IHPFdown-arrow GG 123  ± 12 280  ± 6.7 2.3
hc14     IHPFdown-arrow IHPF 29  ± 4.8 35  ± 6.6 1.2
hc15     IRVYdown-arrow IHPF 5.9  ± 0.1 0.32  ± 0.001 0.054
hc16     DHVYdown-arrow IHPF 12  ± 0.3 0.12  ± 0.001 0.01
hc17     DRPYdown-arrow IHPF 25  ± 0.7 0.15  ± 0.001 0.006
hc18     DRVFdown-arrow IHPF 8.0  ± 0.4 0.028  ± 0.001 0.0035
hc19     IHVYdown-arrow IHPF 13.6  ± 1.2 1.04  ± 0.003 0.071
hc20     DRPFdown-arrow IHPF 20  ± 0.9 0.09  ± 0.001 0.0045
hc21     IHVFdown-arrow IHPF 9.0  ± 2.6 0.7  ± 0.04 0.077
hc22 DRVYIHPFdown-arrow IGGGG 19  ± 1.4 55  ± 1.7 2.9
hc23 DRVYIHPFdown-arrow ethyl ester 19  ± 1.0 1,744  ± 5.0 92

a  For positional nomenclature of residues, see Ref. 16. Arrows indicate the peptide bond hydrolyzed by human chymase under assay conditions.

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 DRVYIHPFdown-arrow HL (Ang I) with that of DRVYIHPFdown-arrow 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 DRVYIHPFdown-arrow G (peptide hc2, also see hc3) occurs with a ~55-fold lower catalytic efficiency than of the Phe8-Gly9 bond in DRVYIHPFdown-arrow 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 (DRVYdown-arrow IHPF; 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 DRVYdown-arrow 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 ESDagger 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 Tyr4down-arrow 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). IHPFdown-arrow HL (peptide hc12) and IHPFdown-arrow 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 IHPFdown-arrow GG (peptide hc13) was ~45-fold higher than for DRVYdown-arrow 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 ESDagger , 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 ESDagger during hydrolysis (Delta Delta GTDagger  = -6.37 kJ·mol-1 for P4 Asp right-arrow Ile, -2.01 kJ·mol-1 for P3 Arg right-arrow His, and -0.67 kJ·mol-1 for P2 Val right-arrow 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 Delta Delta GTDagger . Therefore, with respect to individual component differences between the DRVY and IHPF acyl groups, the P4 Asp right-arrow Ile change has the most favorable effect on reactivity. Additivity analysis indicates a good agreement between the observed [Delta Delta GTDagger (multiple)] and calculated [Sigma Delta Delta GTDagger (components)] decreases in transition state stabilization energy for two- or three-component transitions in the Ang II acyl group, e.g. DRVY right-arrow IHVY, DRVY right-arrow DRPF, or DRVY right-arrow IHVF (peptides hc19-21) (Table II). Remarkably, however, when we compared the observed decrease in transition state stabilization energy associated with the DRVY right-arrow IHPF change in the Ang II acyl group [Delta Delta GTDagger (multiple) = -14.3 kJ·mol-1] with that calculated on the basis of individual P4 to P1 changes [Sigma Delta Delta GTDagger (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 Delta Delta Gbinding (from Km values in Table I) and Delta Delta Gcat (from kcat values in Table I) indicates that for the DRVYdown-arrow IHPF right-arrow IHPFdown-arrow IHPF transition there is good agreement between observed and calculated measurements of Delta Delta Gbinding [Delta Delta Gbinding (multiple) - Sigma Delta Delta Gbinding (components) = 0.95 kJ·mol-1], but deviations from simple additivity are clearly evident in Delta Delta Gcat measurements [Delta Delta Gcat (multiple) - Sigma Delta Delta 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 ESDagger complex but is not observed in the initial energetics of substrate binding that leads to the formation of the ES complex.

Table II.

Contributions of each substrate-human chymase subsite interaction to the catalytic efficiency of peptide substrates hc14, hc19, hc20, and hc21 listed in Table 1 


Substrate pair  Delta Delta GTDagger (P4)  Delta Delta GTDagger (P3)  Delta Delta GTDagger (P2)  Delta Delta GTDagger (P1)  Sigma Delta Delta GTDagger (Pn)a  Delta Delta GTDagger (observed)b

kJ·mol-1
DRVYdown-arrow IHPF right-arrow IHPFdown-arrow IHPF (Ang II right-arrow peptide hc14)  -6.37  -2.01  -0.67 0.712  -8.34  -14.3
DRVYdown-arrow IHPF right-arrow IHVYdown-arrow IHPF (Ang II right-arrow peptide hc19)  -6.37  -2.01  -8.38  -7.08
DRVYdown-arrow IHPF right-arrow DRPFdown-arrow IHPF (Ang II right-arrow peptide hc20)  -0.67 0.712 0.042 0.042
DRVYdown-arrow IHPF right-arrow IHVFdown-arrow IHPF (Ang II right-arrow peptide hc21)  -6.37  -2.01 0.712  -7.67  -7.29

a  Sigma Delta Delta GTDagger (Pn) is the sum of the Delta Delta GTDagger (Xaa right-arrow Yaa) values for the substituted amino acids in substrate pairs. The Delta Delta GTDagger (Xaa right-arrow Yaa) values are obtained from kcat/Km values from Table I for substrate pairs in which only one position is substituted; calculated from peptides pairs Ang II right-arrow peptide hc15 (for P4), Ang II right-arrow peptide hc16 (for P3), Ang II right-arrow peptide hc17 (for P2), and Ang II right-arrow peptide hc18 (for P1).
b  Delta Delta GTDagger (observed A right-arrow B) = -RT ln [kcat/Km(B)/kcat/Km(A)], i.e. the actual difference in transition state stabilization energy of the two substrates A and B.

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 Phe8down-arrow His9 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.

Why Rat Chymase-1 Is an Angiotensinase

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 IHPFdown-arrow HL (peptide rc1). Therefore, we used DRVYdown-arrow IHPF and IHPFdown-arrow 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.

Table III.

Hydrolysis of Ang II and its analogues by rat chymase-1

The kinetic constants were determined in 20 mM Tris-HCl buffer, pH 8.0, containing 0.5 M KCl, and 0.01% Triton X-100 at 37 °C. Km and kcat values were determined by nonlinear regression; values are the means ± S.D. of three independent determinations for each peptide.
Peptide name/ reference number Substrate (Pn, P3, P2, P1<UNL><SUP>↓</SUP></UNL>P'1, P'2, P'3, P'n)a Km kcat kcat/Km

µm s-1 µm-1·s-1
Ang II DRVYdown-arrow IHPF 55  ± 2.7 4.7  ± 0.08 0.085
rc1 IHPFdown-arrow HL 250  ± 7.8 1.08  ± 0.011 0.0043
rc2 IHPFdown-arrow GG 420  ± 6.5 1.7  ± 0.005 0.004
rc3 IHPFdown-arrow GGG 320  ± 5.0 1.6  ± 0.03 0.005
rc4 IHPFdown-arrow GGGG 310  ± 14 2.0  ± 0.02 0.006
rc5 IHPFdown-arrow GGGGG 200  ± 10 1.6  ± 0.03 0.008
rc6 DRVYdown-arrow GG 180  ± 5.0 0.7  ± 0.04 0.0039
rc7 DRVYdown-arrow GHPF 54  ± 3.0 0.71  ± 0.004 0.013
rc8 DRVYdown-arrow IGPF 35  ± 0.4 14  ± 0.08 0.4
rc9 DRVYdown-arrow IHGF 25  ± 2.1 8.0  ± 0.11 0.32
rc10 DRVYdown-arrow IHPG 58  ± 2.0 33  ± 0.21 0.56
rc11 IHPFdown-arrow IHPF 9.0  ± 1.0 7.5  ± 0.1 0.83
rc12 IHPFdown-arrow IGGGG 16  ± 0.1 8.4  ± 0.1 0.53
rc13 IHPFdown-arrow IGIGG 14  ± 0.86 6.9  ± 0.38 0.49
rc14 IHPFdown-arrow IGIGI 14  ± 2.2 7.8  ± 0.07 0.55

a  For positional nomenclature of residues, see Ref. 16. Arrows indicate the peptide bond hydrolyzed by rat chymase-1 under assay conditions.

No difference in the catalytic efficiency was observed between IHPFdown-arrow HL (peptide rc1) and IHPFdown-arrow 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). DRVYdown-arrow GG (peptide rc6) cleavage by rat chymase-1 occurs with a ~22-fold lower catalytic efficiency than DRVYdown-arrow 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 P'1 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 [Sigma Delta Delta GTDagger (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 [Delta Delta GTDagger (P'1) = -4.84 kJ·mol-1] to generate an ~7.5 kJ·mol-1 increase in the free energy required to reach the ESDagger complex during hydrolysis (Table IV). However, the overall observed effect of Ang II leaving group interactions on catalytic efficiency is favorable (Delta Delta GTDagger  = -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 DRVYdown-arrow 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.

Table IV.

Contributions of leaving group substrate residues-rat chymase-1 subsite interactions to the catalytic efficiency of peptide substrates rc6, rc7, rc8, rc9, rc10, and rc11 listed in Table III


Substrate pair  Delta Delta GTDagger a

kJ·mol-1
DRVYdown-arrow IHPF right-arrow DRVYdown-arrow GHPF (Ang II right-arrow peptide rc7) 4.84
DRVYdown-arrow IHPF right-arrow DRVYdown-arrow IGPF (Ang II right-arrow peptide rc8)  -4.0
DRVYdown-arrow IHPF right-arrow DRVYdown-arrow IHGF (Ang II right-arrow peptide rc9)  -3.42
DRVYdown-arrow IHPF right-arrow DRVYdown-arrow IHPG (Ang II right-arrow peptide rc10)  -4.86
DRVYdown-arrow GG right-arrow DRVYdown-arrow IHPF (peptide rc6 right-arrow Ang II)  -7.94
IHPFdown-arrow GG right-arrow IHPFdown-arrow IHPF (peptide rc2 right-arrow peptide rc11)  -13.8

a  Delta Delta GTDagger (A right-arrow B) = -RT ln [kcat/Km(B)/kcat/Km(A)], i.e. difference in transition state stabilization energy of the two substrates A and B.

Direct comparisons were made between the Ang II acyl group DRVY and the Ang I acyl group IHPF. Specificity constants for the cleavage of IHPFdown-arrow GG (peptide rc2) and DRVYdown-arrow 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 Delta Delta GTDagger when DRVY is the acyl group and a -13.8 kJ·mol-1 decrease in Delta Delta GTDagger 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 IHPFdown-arrow IHPF catalysis can be mimicked by the introduction of an Ile side chain at the P'1 position, we examined IHPFdown-arrow IHPF (peptide rc11), IHPFdown-arrow GGGG (peptide rc4), IHPFdown-arrow IGGGG (peptide rc12), and IHPFdown-arrow GGGGG (peptide rc5) catalysis by rat chymase-1. IHPFdown-arrow IHPF was cleaved by rat chymase-1 with a 138-fold higher catalytic efficiency than IHPFdown-arrow GGGG. IHPFdown-arrow IGGGG was cleaved with a 66-fold higher catalytic efficiency than IHPFdown-arrow GGGGG; peptides in which Ile side chains were introduced at additional leaving group sites, e.g. IHPFdown-arrow IGIGG and IHPFdown-arrow IGIGI (peptides rc13 and rc14), were catalyzed with an efficiency similar to that of IHPFdown-arrow 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 P'1 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 beta -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.

General Considerations about the S and the S' Subsites of Human Chymase and Rat Chymase-1

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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL44201. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Molecular Cardiology, FF30, Research Inst., The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-2057; Fax: 216-444-9410; E-mail: husaina{at}cesmtp.ccf.org.
1    Mast cell chymotrypsin-like proteases have in the past been variously referred to as chymases, mast cell chymases, and mast cell proteases. The term mast cell protease has also been used to designate mouse mast cell tryptases and carboxypeptidases; thus, not all mouse enzymes referred to as mast cell proteases are chymases; the number designation associated with this nomenclature is usually 1, 2, etc., but L has also been used. The number assignment has usually been given in order of discovery. Because homologs in different species have been discovered in differing orders, numbers do not necessarily correspond to homologs. In this paper, we have used the term chymase to describe a distinct group of leukocyte serine proteases (7) but have retained the original number designation given at the time of discovery.
2    The abbreviations used are: Ang I, angiotensin I; Ang II, angiotensin II; down-arrow , scissile bond in peptide substrates; HPLC, high pressure liquid chromatography; ES complex, enzyme-substrate complex; ESDagger complex, transition state complex; Delta Delta G7Dagger , difference between two substrates in the free energy required for transition state stabilization (i.e. free energy required to reach ESDagger complex from E + S.
3    The nomenclature used for the individual amino acids (P1, P'1, etc.) of a substrate and the subsites (S1, S'1, etc.) of the enzyme is that of Schechter and Berger (21). Amino acid residues of substrates numbered P1, P2, etc. are toward the N-terminal direction, and P'1, P'2, etc. are toward the C-terminal direction from the scissile bond.
4    Our interpretation of the these and other kinetic data described in this paper is based on the arguments presented by Fersht (15) in his discussions of enzyme-substrate complementarity and the use of binding energy in catalysis. Also germane to these discussions is the commonly held view that for serine protease-dependent amide bond cleavage, the rate-determining step is the acylation step (k2 is the acylation rate constant), whereas for ester bond cleavage, the deacylation step is rate-limiting (k3 is the deacylation rate constant). However, recent studies suggest that some amide bonds in substrates with highly efficient acyl groups are hydrolyzed by leukocyte serine proteases with rate-limiting deacylation (11, 22). We found that the kcat for human chymase-dependent Ang II-ethyl ester (peptide hc23) and Ang I cleavage is 1,740 ± 5.0 s-1 and 146 ± 3.3 s-1 (in each case n = 3), respectively. Because k2 >>  k3 for ester bond cleavage, the rate constant k3 for the deacylation of the human chymase-Ang II complex will be equal to kcat. Thus, using the formula 1/kcat = 1/k2 + 1/k3, k2 is ~160 s-1 for human chymase-dependent conversion of Ang I to Ang II. We speculate that in these highly efficient human chymase reactions, kcat adequately reflects k2 and hence is a measure of enzyme-transition state complementarity.

Acknowledgments

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.


REFERENCES

  1. Brain, S. D., and Williams, T. J. (1988) Nature 335, 73-75 [CrossRef][Medline] [Order article via Infotrieve]
  2. Woodbury, R. G., Miller, H. R., Huntley, J. F., Newlands, G. F., Palliser, A. C., and Wakelin, D. (1984) Nature 312, 450-452 [Medline] [Order article via Infotrieve]
  3. Le Trong, H., Neurath, H., and Woodbury, R. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 364-367 [Abstract]
  4. Caughey, G. H., Leidig, F., Viro, N. F., and Nadel, J. A. (1988) J. Pharmacol. Exp. Ther. 244, 133-137 [Abstract]
  5. Urata, H., Kinoshita, A., Misono, K. S., Bumpus, F. M., and Husain, A. (1990) J. Biol. Chem. 265, 22348-22357 [Abstract/Free Full Text]
  6. Benditt, E. P., and Arase, M. (1959) J. Exp. Med. 110, 451-460
  7. Chandrasekharan, U. M., Sanker, S., Glynias, M. J., Karnik, S. S., and Husain, A. (1996) Science 271, 502-505 [Abstract]
  8. Itoh, H., Murakumo, Y., Tomita, M., Ide, H., Kobayashi, T., Maruyama, H., Horii, Y., and Nawa, Y. (1996) Biochem. J. 314, 923-929 [Medline] [Order article via Infotrieve]
  9. Urata, H., Boehm, K. D., Philip, A., Kinoshita, A., Gabrovsek, J., Bumpus, F. M., and Husain, A. (1993) J. Clin. Invest. 91, 1269-1281 [Medline] [Order article via Infotrieve]
  10. Hoit, B. D., Shao, Y., Kinoshita, A., Gabel, M., Husain, A., and Walsh, R. A. (1995) J. Clin. Invest. 95, 1519-1527 [Medline] [Order article via Infotrieve]
  11. Powers, J. C., Tanaka, T., Harper, J. W., Minematsu, Y., Barker, L., Lincoln, D., Crumley, K. V., Fraki, J. E., Schechter, N. M., Lazarus, G. G., Nakajima, K., Nakashino, K., Neurath, H., and Woodbury, R. G. (1985) Biochemistry 24, 2048-2058 [Medline] [Order article via Infotrieve]
  12. Kinoshita, A., Urata, H., Bumpus, F. M., and Husain, A. (1991) J. Biol. Chem. 266, 19192-19197 [Abstract/Free Full Text]
  13. Wells, J. A. (1990) Biochemistry 29, 8509-8517 [Medline] [Order article via Infotrieve]
  14. Polgar, L. (1989) Mechanisms of Protease Action, pp. 87-122, CRC Press, Boca Raton, FL
  15. Fersht, A. R. (1985) Enzyme Structure and Mechanism, 2nd Ed., pp. 311-344, W. H. Freeman, New York
  16. Coombs, G. S., Dang, A. T., Madison, E. L., and Corey, D. R. (1996) J. Biol. Chem. 271, 4461-4467 [Abstract/Free Full Text]
  17. Schellenberger, V., Braune, K., Hofmann, H., and Jakubke, H. (1991) Eur. J. Biochem. 199, 623-636 [Abstract]
  18. Gron, H., and Breddam, K. (1992) Biochemistry 31, 8967-8971 [Medline] [Order article via Infotrieve]
  19. Schellenberger, V., Turck, C. W., and Rutter, W. J. (1994) Biochemistry 33, 4251-4257 [Medline] [Order article via Infotrieve]
  20. Bastos, M., Maeji, N. J., and Abeles, R. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6738-6742 [Abstract]
  21. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162 [Medline] [Order article via Infotrieve]
  22. Stein, R. L., Viscarello, B. R., and Wildonger, R. A. (1984) J. Am. Chem. Soc. 106, 796-798

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.