Evidence for the negative cooperativity of the two active sites within bovine somatic angiotensin-converting enzyme

Abstract

The somatic isoform of angiotensin-converting enzyme (ACE) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. We have used the two-domain ACE form and its individual domains to compare characteristics of different domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz-Phe-His-Leu ( N-carbobenzoxy- L-phenylalanyl- L-histidyl- L-leucine; from the panel of seven) was hydrolyzed faster by the N-domain, the substrates FA-Phe-Gly-Gly ( N-(3-[2-furyl]acryloyl)- L-phenylalanyl-glycyl-glycine) and Hip-His-Leu ( N-benzoyl-glycyl- L-histidyl- L-leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C-domain. The inhibitor captopril ((2 S)-1-(3-mercapto-2-methylpropionyl)- L-proline) bound to the N-domain more effectively than to the C-domain, whereas lisinopril (( S)- N α-(1-carboxy-3-phenylpropyl)- L-lysyl- L-proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two-domain or single-domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k cat values obtained for somatic enzyme was the sum of k cat values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two-domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single-domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity.