Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

Abstract

The interaction of angiotensin converting enzyme with 6 metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N.alpha.-(3-mercaptopropanoyl)-L-Arg, N.alpha.-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and 3 dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots varied with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while 5 of 7 tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp and MK-422 were 8.3-, 5.5-, 4.7- and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent. By curve fitting, these plots were shown to be consistent with a rate equation of the form v/[E0] = (1 + d[I])/(a + b[I] + c[I]2), suggesting that inhibitor can bind to more than one enzyme form and that there are alternative pathways to product. Two inhibition mechanisms are described which incorporate these features and may account for the observed substrate dependence. These mechanisms attribute the unusual kinetics inhibitor binding to an enzyme-product complex or interaction of the enzyme with activating anions. A 3rd mechanism, consistent with the kinetic observations, involves multiple inhibitor binding and appears unlikely on the basis of equilibrium dialysis measurements.