Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid

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Abstract
Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25.degree. C by 3-bromopropionic acid [3-BPA]. In the absence of NAD+ , the Ki is 2 nM and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM and k3/Ki is 0.006 M-1 s-1. 3-BPA is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+.cntdot.pyrazole and NADH.cntdot.isobutyramide, was retarded by NAD+, NADH or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+ or NADH. Enzyme was inactivated with radioactive 3-BPA, aminoethylated and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-BPA with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the Zn at the active site. Carboxyethylation by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.