Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart

• Published in: The Journal of biological chemistry Vol. 266; no. 29; pp. 19192 - 19197
• Main Authors: KINOSHITA, A, URATA, H, BUMPUS, F. M, HUSAIN, A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.10.1991
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; angiotensin I; Angiotensin II - metabolism; Biological and medical sciences; Chymases; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; heart; Humans; Hydrolases; Hydrolysis; Kinetics; Molecular Sequence Data; Myocardium - enzymology; Neurotensin - metabolism; Sequence Homology, Nucleic Acid; Serine Endopeptidases - metabolism; Substrate Specificity; Heart; Human; Site specificity; Purification; Enzyme; Substrate specificity; Chymosin; Peptide hormone; Binding site; Kinetic parameter; Angiotensin II
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)54981-4

Human heart chymase, a chymotrypsin-like serine proteinase that hydrolyzes the Phe8-His9 bond in angiotensin I (Ang I) to yield the octapeptide hormone angiotensin II (Ang II) and His-Leu, is the most specific, efficient Ang II-forming enzyme described. Other mammalian chymases display a much broader substrate specificity. To better define its substrate specificity, we have mapped the extended substrate-binding site of human heart chymase using Ang I analogs. The enzyme has a preference for aromatic amino acids phenylalanine, tyrosine, and tryptophan at the P1 site. At the S2 subsite there is a significant preference for proline over hydrophobic or hydrophilic amino acids. There is no clear preference for hydrophobic or hydrophilic amino acids at the S'1 and S'2 subsites, but an Ang I analog containing a P'1 proline is not hydrolyzed and one with a P'2 proline is hydrolyzed poorly. An increasing reduction in reactivity occurs when the P position amino acids in Ang I are deleted sequentially from the N terminus. An increase or decrease in the length of the His-Leu leaving group also produces a marked decrease in reactivity. No single determinant in Ang I is preeminently required for efficient catalysis, but several factors acting synergistically appear to be important. Thus, we propose that ideal substrates for human heart chymase should contain the structure nXaa-Pro-[Phe, Tyr, or Trp]-Yaa-Yaa, where n greater than or equal to 6; Xaa = any amino acid; Yaa = any amino acid except proline. This structure exists in Ang I and neurotensin, both of which are good substrates for human heart chymase. These findings indicate that the selection of the scissile bond by the extended substrate-binding site of human heart chymase is more restricted than that in other chymases.