HUMAN ACYLPEPTIDE HYDROLASE - STUDIES ON ITS THIOL-GROUPS AND MECHANISM OF ACTION

The presence of a cysteine residue(s) near the active site of acylpept ide hydrolase was suggested by inactivation of the enzyme with sulfhyd ryl-modifying agents and by the substantial protection against inactiv ation afforded by the competitive inhibitor acetylmethionine. 5,5'-dit hiobis-(2-nitrobenzoate) titrations of the native and the denatured en zyme together with analysis for cysteic acid after performic acid oxid ation showed that the enzyme contained 12 free SH groups and three dis ulfide bonds/monomer. Chemical modification with radiolabeled iodoacet amide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barri er to the efficient diffusion of substrates into and products out of t he active site. Studies on the mechanism of action of acylpeptide hydr olase have employed p-nitrophenyl-N-propyl carbamate as a potent activ e site-directed inhibitor. Enzyme inactivation, which follows pseudo f irst-order kinetics, is diminished by the competitive inhibitor acetyl methionine. The inhibited enzyme slowly regains activity at a rate tha t is increased in the presence of the nucleophile hydroxylamine. A gen eral mechanism involving an acyl enzyme intermediate is supported by e vidence for the formation of acetyl-alanyl hydroxamate during hydrolys is of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effects on V-max and K-m during this reaction indicate that hydrol ysis of the acyl-enzyme intermediate is rate limiting.