Escherichia coli L-threonine dehydrogenase is a member of the Zn(2+)-containing alcohol/polyol dehydrogenase family. Methylation of His-90 of L-threonine dehydrogenase was recently found to cause total inactivation (J. P. Marcus and E. E. Dekker, 1995 Arch. Biochem. Biophys. 316, 413-420). Since His-90 is not conserved among the related dehydrogenases, this residue was changed to arginine, asparagine, and alanine by site-directed mutagenesis in order to probe its role. All three purified, homogeneous mutants, like wild-type enzyme, contained one Zn2+ atom/subunit and exhibited a sequential catalytic mechanism; the kcat value for each, however, was reduced approximately 10-fold. The K(m) value for threonine was elevated from 3 mM for wild-type enzyme to 31, 328, and 417 mM, respectively, for mutants H90R, H90N, and H90A. The activation energy of catalysis for mutant H90A was increased by 6.6 kcal/mol, suggesting that in the wild-type enzyme His-90 forms at least one crucial hydrogen bond in the transition state. Whereas wild-type enzyme catalyzed the oxidation of threonine amide (0.75 M) about twice as fast as this same concentration of threonine or 0.375 M L-2-amino-3-hydroxypentanoate, the reaction rate of mutant H90A with 0.75 M threonine amide or threonine methyl ester was 33- to 35-fold higher than with this level of threonine. Similarly, mutant H90N used 0.75 M threonine methyl ester or threonine amide as substrate 9- to 13-fold better than it used this concentration of threonine. Mutants H90A and H90N were more reactive with 0.225 M L-threonine hydroxamate than with 0.75 M threonine, but mutant H90A did not oxidize L-2-amino-3-hydroxypentanoate (0.375 M) and mutant H90N used this substrate poorly. The best substrates for mutant H90R were threonine methyl ester, threonine, and threonine amide (all tested at 0.75 M); 0.375 M L-2-amino-3-hydroxypentanoate was a poor substrate. The isolation and characterization of these first His-90 mutants of E. coli L-threonine dehydrogenase confirm the importance of this residue in catalysis and suggest that His-90 is an active-site residue which modulates the substrate specificity of L-threonine dehydrogenase.