Identification of a second 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 methyl p-nitrobenzenesulfonate results in a time- and concentration-dependent loss of enzymatic activity. As the concentration of the methylating agent is increased, the rate of inactivation reaches a limiting value of 0.01 min-1 at pH 7.0 and 25 degrees C, suggesting that the inhibitor is binding at a specific site prior to reaction. Approximately one methyl group is incorporated per enzyme subunit inactivated. Reaction with [14C]methyl p-nitrobenzenesulfonate followed by amino acid analysis 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; the radioactivity is associated with histidine residue-90. The Zn2+ content of the inactivated and the native enzyme remains the same. The substrate, L-threonine, and substrate analogs, L-threonine methyl ester and L-threonine amide, provide about 60% protection against inactivation, whereas NAD+ has no effect. In contrast, NADH markedly enhances the rate of inactivation by this methylating agent, suggesting a possible conformational change in the vicinity of His-90 is induced by binding of the coenzyme.