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Volume 272, Number 5, Issue of January 31, 1997 pp. 2963-2968
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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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)

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 (k_cat/K_m) 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 P₁ Ile⁵. The overall effect of this P₁ 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 P₁ 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.

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