IDENTIFICATION OF A 2ND ACTIVE-SITE RESIDUE IN ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE - METHYLATION OF HISTIDINE-90 WITH METHYL P-NITROBENZENESULFONATE

Incubation of L-threonine dehydrogenase from Escherichia coli with met hyl p-nitrobenzenesulfonate results in a time- and concentration-depen dent loss of enzymatic activity, As the concentration of the methylati ng agent is increased, the rate of inactivation reaches a limiting val ue of 0.01 min(-1) at pH 7.0 and 25 degrees C, suggesting that the inh ibitor is binding at a specific site prior to reaction. Approximately one methyl group is incorporated per enzyme subunit inactivated. React ion with [C-14]methyl p-nitrobenzenesulfonate followed by amino acid a nalysis shows that greater than 90% of the radioactivity incorporated into the enzyme is associated with a peak that coelutes with 3-methyl- N-histidine. Tryptic digestion of the inactive enzyme adduct yields a radioactive peptide corresponding to residues 85-97 of the protein; th e radioactivity is associated with histidine residue-90. The Zn2+ cont ent of the inactivated and the native enzyme remains the same, The sub strate, L-threonine, and substrate analogs, L-threonine methyl ester a nd L-threonine amide, provide about 60% protection against inactivatio n, whereas NAD(+) has no effect. In contrast, NADH markedly enhances t he rate of inactivation by this methylating agent, suggesting a possib le conformational change in the vicinity of His-90 is induced by bindi ng of the coenzyme. (C) 1995 Academic Press, Inc.