title: Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid.
abstract: Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, 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-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X 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-bromopropionic acid, 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-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.
mesh_terms: D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D003545:Cysteine; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011422:Propionates / Q000494:pharmacology*
chemical_list: D010446:Peptide Fragments; D011422:Propionates; D009243:NAD; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D003545:Cysteine; C035776:3-bromopropionic acid