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

• Published in: The Journal of biological chemistry 1997-01, Vol.272 (5), p.2963-2968
• Main Authors: Sanker, Subramaniam, Chandrasekharan, Unnikrishnan M., Wilk, Dennis, Glynias, Manuel J., Karnik, Sadashiva S., Husain, Ahsan
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Angiotensin II - metabolism; Animals; Binding Sites; Chymases; Heart Ventricles; Humans; Kinetics; Mast Cells - enzymology; Myocardium - enzymology; Oligopeptides - chemistry; Oligopeptides - metabolism; Rats; Regression Analysis; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Substrate Specificity; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.5.2963

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.