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.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
J P
JP
Marcus
E E
EE
Dekker
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001557:Benzenesulfonates / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D008745:Methylation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013912:Threonine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D001557:Benzenesulfonates; D009243:NAD; D013912:Threonine; D006639:Histidine; C010233:methyl-4-nitrobenzenesulfonate; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase