Lys40 but not Arg143 influences selectivity of angiotensin conversion by human α-chymase

Abstract

Human α-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe⁸ to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat β-chymase 1, are much less selective, destroying AT by cleaving at Tyr⁴. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys⁴⁰ or Arg¹⁴³ accounts for the human enzyme’s marked preference for Phe⁸ over Tyr⁴. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys⁴⁰ or Arg¹⁴³ to neutral residues. Lys⁴⁰ was exchanged for alanine, the residue found in canine α- and rat β-chymase 1, the latter being dramatically less selective for hydrolysis at Phe⁸. Arg¹⁴³ was exchanged for glutamine found in rat β-chymase 1. The Lys⁴⁰Ala mutant is a dog-like enzyme retaining strong preference for Phe⁸ but with Tyr⁴ hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg¹⁴³Gln mutant, contrary to prediction, remains highly Phe⁸-selective. Therefore, Lys⁴⁰, but not Arg¹⁴³, contributes to human chymase’s remarkable preference for AT II generation over destruction.