Identification of a Second Active Site Residue in Escherichia coli L-Threonine Dehydrogenase: Methylation of Histidine-90 with Methyl p-Nitrobenzenesulfonate

• Published in: Archives of biochemistry and biophysics Vol. 316; no. 1; pp. 413 - 420
• Main Authors: Marcus, J.P., Dekker, E.E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-01-1995
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - metabolism; Amino Acid Sequence; Benzenesulfonates - chemistry; Binding Sites; Escherichia coli - enzymology; Histidine - chemistry; Methylation; Models, Chemical; Molecular Sequence Data; NAD - pharmacology; Oxidation-Reduction; Peptide Mapping; Sequence Homology, Amino Acid; Stereoisomerism; Threonine - analogs & derivatives; Threonine - metabolism; Threonine - pharmacology
• DOI: 10.1006/abbi.1995.1055
• EISSN: 1096-0384
• ISSN: 0003-9861

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°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.