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

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 P 1 and P 2 preferences, which are the major determinants of specificity. Surprisingly, despite the occurrence of optimal P 2 and P 1 residues at the Phe 82 . 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 . Early comparative studies on chymase specificities by Powers et al. (11) using peptide 4-nitroanilide substrates indicated that the S 1 to S 4 subsites 3 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 P 1 hydrophobic aromatic residue, a P 2 hydrophobic residue or Pro, and a P 3 hydrophobic residue. S 4 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 S 1 to S 4 subsites of these enzymes could be important for determining specificity. In a previous paper we explored the S 1 subsite as well as the SЈ 1 to SЈ 2 subsites of human chymase using decapeptide Ang I analogs. We showed that a P 1 hydrophobic aromatic residue was necessary but that several non-* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed: Dept. of Molecular Cardiology, FF30,...

Involvement of chymase-mediated angiotensin II generation in blood pressure regulation

“…r-c-myc-tagged mouse chymases cleave these substrates at a single site (indicated by the arrow). Conversion of Ang I-(5-10) to Ang II-(5-8) is expected to reflect the potential of the chymases to convert Ang I to Ang II, because the P 4 to P 1 and P 1 ′ to P 2 ′ residues of the substrate are known to be crucial in determining catalytic specificity (15,16). In Ang I-(5-10) and Ang I, the substrate P 4 to P 1 residues (i.e., IHPF) and P 1 ′ to P 2 ′ residues (i.e., HL) are identical.…”

Albumin Is a Substrate of Human Chymase

“…It also should be emphasized that any non-additive interactions, favorable or unfavorable, between substrate residues occupying different subsite positions will tend to make an individual substrate appear more or less acceptable in comparison to the suitability predicted from mixtures of peptides, as in our study. The possibility of such interactions between subsites is suggested by a study of a library of non-cleavable, peptidic inhibitors of chymase (40) as well as by studies of chymase-mediated hydrolysis of angiotensin analogs (27). The observed effect of The adjacent lane contains HSA incubated in buffer alone.…”

“…This side chain is aromatic for chymases and other chymotryptic enzymes. In the case of chymase-mediated hydrolysis of angiotensin I, interactions involving P1, P1Ј, and P2Ј are particularly important (24,25,27,28). However, for many serine peptidases (including chymase and cathepsin G hydrolyzing other peptide substrates), interactions on the other side of P1 (involving P4, P3, and P2) critically influence substrate binding and hydrolysis (29,30).…”

Effect of Angiotensin II Type 1 Receptor Blocker on Cardiac Angiotensin-Converting Enzyme and Chymase-Like Activities, and Cardiac Fibrosis in Cardiomyopathic Hamsters

“…Differences between rats and dogs can be caused by the cardiac Ang II-forming mechanism and the effect of cardiac chymase. In the rat heart, Ang II is mainly produced by cardiac ACE, not by cardiac chymase, and is degraded by cardiac chymase [16].Because of the difference in cardiac Ang II-forming pathways by species, hamsters were used in this study to examine the effect of AT1 receptor blockers on cardiac fibrosis in dogs. After 4 weeks of administration of an AT1 receptor blocker to the hamsters, echocardiography, assessment of cardiac Ang II-forming enzymes, and histological examination of the heart were conducted to investigate the relationships between cardiac RAS and cardiac function/ remodeling.…”

“…Differences between rats and dogs can be caused by the cardiac Ang II-forming mechanism and the effect of cardiac chymase. In the rat heart, Ang II is mainly produced by cardiac ACE, not by cardiac chymase, and is degraded by cardiac chymase [16].…”