Methylation of the active center histidine 217 in D-amino acid oxidase by methyl-p-nitrobenzenesulfonate.

Incubation of D-amino acid oxidase with excess methyl-p-nitrobenzenesulfonate results in a pseudo-first order, irreversible loss of 95% of the assayable activity using D-phenylglycine as substrate. The rate of inactivation reaches a limiting value of 0.021 min-1 (pH 7.7, 22 degrees C) as the concentration of inhibitor is increased, consistent with a two-step mechanism with complex formation prior to covalent inactivation. This rate decreases with decreasing pH in a manner that suggests that the basic form of a group within the enzyme, with an apparent pKa of 6.7, is required for inactivation. Approximately three [14C]methyl residues are covalently incorporated at maximal inactivation. The competitive inhibitor benzoate prevents inactivation and the incorporation of approximately 1 methyl residue. Histidine 217, the residue modified by 5-dimethylaminonaphthalene-1-sulfonyl chloride (Swenson, R. P., Williams, C. H., Jr., and Massey, V. (1983) J. Biol. Chem. 258, 497-502), was identified as the major residue modified in the absence of benzoate. Both the 1-methyl and 3-methyl derivatives of histidine 217 were observed in a mole ratio of 0.64:0.36.