Inhibition of angiotensin converting enzyme: dependence of chloride

Abstract

Activation of angiotensin converting enzyme (ACE) by Cl- is strongly dependent on substrate structure, and 3 substrate classes have been identified on the basis of activation behavior. The present study examines the Cl- dependence of the inhibition of ACE by 9 inhibitors [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), L-Ala-L-Pro, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-L-Trp, N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, L-Phe-L-Arg, N.alpha.-(3-mercaptopropanoyl)-L-Arg and N.alpha.-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Lys] containing structural features characteristic of the 3 classes of substrates. Apparent Ki values for all inhibitors are markedly (70- to 250-fold) decreased by 300 mM Cl-. However, the enhancement of inhibition is achieved at significantly lower Cl- concentrations with those inhibitors having an ultimate arginine or lysine than with the remainder. This variability parallels that previously found for activation of substrate hydrolysis. The effect of Cl- on the individual steps in the formation and dissociation of the steady-state enzyme-inhibitor complexes was determined with the slow-binding inhibitor MK-422. Pre-steady-state analysis indicates that binding of both MK-422 and captopril follows a (minimally) 2-step mechanism: .**GRAPHIC**. in which rapid formation of an enzyme-inhibitor complex is followed by a slow isomerization. In the presence of 300 mM Cl-, the calculated values for k3, k4, Ki, and the overall inhibition constant (Ki*) with MK-422 are 1.9 .times. 10-2 s-1, 1.1 .times. 10-4 s-1, 9.2 .times. 10-9 M, and 5.0 .times. 10-11 M, respectively. In the presence of 20 mM Cl-, k3, k4, Ki, and Ki* values are 8.7 .times. 10-3 s-1, 5.4 .times. 10-4 s-1, 6.3 .times. 10-9 M, and 3.7 .times. 10-10 M, respectively. Thus, Cl- influences isomerization, not initial complex formation, and serves largely to stabilize EI* and thereby slow the conversion of EI* to EI.