Observation of a chloride-dependent intermediate during catalysis by angiotensin converting enzyme using radiationless energy transfer

Publisher Website
Google Scholar
Abstract
Stopped-flow radiationless energy-transfer kinetics have been used to examine the effects of chloride on the hydrolysis of Dns-Lys-Phe-Ala-Arg by angiotensin converting enzyme. The kinetic constants for hydrolysis at pH 7.5 and 22.degree. C in the presence of 300 mM sodium chloride were KM = 28 .mu.M and kcat = 110 s-1, and its absence Km = 240 .mu.M and Kcat = 68 s-1. The apparent binding constant for chloride was 4 mM, and the extent of chloride activation in terms of kcat/KM was 14-fold. The effects of chloride on the pre-steady were examined at 2.degree. C. In the presence of chloride, two distinct enzyme-substrate complexes were observed, suggesting multiple steps in substrate binding. The initial complex was formed during the mixing period (kobsd > 200 s-1) while the second complex was formed much more slowly (kobsd = 40 s-1 when [S] = 5 .mu.M and [NaCl] = 150 mM). Strikingly, in the absence of chloride, only a single, rapidly formed enzyme-substrate complex was observed. These results are consistent with a nonessential activator kinetic mechanism in which the slow step reflects conversion of an initially formed complex, (E.cntdot.Cl-.cntdot.S)1, to a more tightly bound complex, (E.cntdot.Cl-.cntdot.S)2.