Lys40 but not Arg143 influences selectivity of angiotensin conversion by human alpha-chymase.
Human alpha-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 beta-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 alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-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.
Muilenburg
Diego J
DJ
Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, 90 Medical Center Way, Box 0911, San Francisco, CA 94143-0911, USA.
Raymond
Wilfred W
WW
Wolters
Paul J
PJ
Caughey
George H
GH
D000809:Angiotensins / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D053818:Chymases; D004285:Dogs; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D000809:Angiotensins; D012697:Serine Endopeptidases; D053818:Chymases