Distinct multisite synergistic interactions determine substrate specificities of human chymase and rat chymase-1 for angiotensin II formation and degradation

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(8 down arrow) and Tyr(4 down arrow) bonds (where down arrow, indicates the scissile bond in peptide substrates) in Ang I (DRVYIHPFHL), human chymase cleaves the Phe(8 down arrow) bond with an similar to 750-fold higher catalytic efficiency (k(cat)/K-m) than the Tyr(4 down arrow) bond in Ang II (DRVYIHPF), whereas rat chymase-1 cleaves the Tyr(4 down arrow) bond with an similar to 20-fold higher catalytic efficiency than the Phe(8 down arrow) bond. Differences in the acyl groups IHPF and DRVY at the Phe(8 down arrow) and Tyr(4 down arrow) bonds, respectively, are chiefly responsible for the preference of human chymase for the Phe(8 down 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 Tyr(4 down arrow) bond, mainly because of a catalytically productive interaction between the enzyme and the P'(1), Ile(5). 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.