SITE-DIRECTED MUTAGENESIS OF HISTIDINE-90 IN ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE ALTERS ITS SUBSTRATE-SPECIFICITY

Escherichia coli L-threonine dehydrogenase is a member of the Zn2+-con taining alcohol/polyol dehydrogenase family. Methylation of His-90 of L-threonine dehydrogenase was recently found to cause total inactivati on (J. P. Marcus and E. E. Dekker, 1995 Arch. Biochem. Biophys. 316, 4 13-420). Since His-90 is not conserved among the related dehydrogenase s, this residue was changed to arginine, asparagine, and alanine by si te-directed mutagenesis in order to probe its role. All three purified , homogeneous mutants, like wildtype enzyme, contained one Zn2+ atom/s ubunit and exhibited a sequential catalytic mechanism; the k(ent) valu e for each, however, was reduced similar to 10-fold. The K-m value for threonine was elevated from 3 mM for wildtype 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/mel, su ggesting that in the wild-type enzyme His-90 forms at least one crucia l hydrogen bond in the transition state, Whereas wild-type enzyme cata lyzed the oxidation of threonine amide (0.75 RI) about twice as fast a s this same concentration of threonine or 0.375 RI L-2-amino-3-hydroxy pentanoate, the reaction rate of mutant H90A with 0.75 M threonine ami de or threonine methyl ester was 33- to 35-fold higher than with this level of threonine. Similarly, mutant H90N used 0.75 M threonine methy l ester or threonine amide as substrate 9- to 13-fold better than it u sed this concentration of threonine. Mutants H90A and H90N were more r eactive with 0.225 M L-threonine hydroxamate than with 0.75 M threonin e, but mutant H90A did not oxidize L-2-amino-3-hydroxypentanoate (0.37 5 M) and mutant H90N used this substrate poorly, The best substrates f or mutant H90R were threonine methyl ester, threonine, and threonine a mide (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 H is-90 mutants off. coli L-threonine dehydrogenase confirm the importan ce of this residue in catalysis and suggest that His-90 is an active-s ite residue which modulates the substrate specificity of L-threonine d ehydrogenase. (C) 1998 Academic Press.