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

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe(8) to generate bioactive AT II which can promote cardiac hypertrophy, vascular stenosis', and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr(4). Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys(40) or Arg(143) accounts for the human enzyme's marked preference for Plies over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys(40) or Arg(143) to neutral residues. Lys(40) was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe(8). Arg(143) was exchanged for glutamine found in rat beta-chymase 1. The Lys(40)Ala mutant is a dog-like enzyme retaining strong preference for Plies 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 Arg(143)Gln mutant, contrary to prediction, remains highly Phe(8)-selective. Therefore, Lys(40), but not Arg(143) contributes to human chymase's remarkable preference for AT II generation over destruction. (C) 2002 Elsevier Science B.V. All rights reserved.