Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart.
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.
Kinoshita
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Department of Heart and Hypertension Research, Research Institute of the Cleveland Clinic Foundation, Ohio 44195-5069.
Urata
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Bumpus
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Husain
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D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism*; D053818:Chymases; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009496:Neurotensin / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D000804:Angiotensin II; D009496:Neurotensin; D012697:Serine Endopeptidases; D053818:Chymases