ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 351, No. 1, March 1, pp. 8–16, 1998 Article No. BB970501 Site-Directed Mutagenesis of Histidine-90 in Escherichia coli L-Threonine Dehydrogenase Alters Its Substrate Specificity 1 Adam R. Johnson 2 and Eugene E. Dekker 3 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606 Received July 7, 1997, and in revised form October 23, 1997 more reactive with 0.225 M L-threonine hydroxamate than with 0.75 M threonine, but mutant H90A did not Escherichia coli L-threonine dehydrogenase is a oxidize L-2-amino-3-hydroxypentanoate (0.375 M) and member of the Zn 2/ -containing alcohol/polyol dehy- mutant H90N used this substrate poorly. The best sub- drogenase family. Methylation of His-90 of L-threonine strates for mutant H90R were threonine methyl ester, dehydrogenase was recently found to cause total inac- threonine, and threonine amide (all tested at 0.75 M); tivation (J. P. Marcus and E. E. Dekker, 1995 Arch. Bio- 0.375 M L-2-amino-3-hydroxypentanoate was a poor chem. Biophys. 316, 413–420). Since His-90 is not con- substrate. The isolation and characterization of these served among the related dehydrogenases, this resi- first His-90 mutants of E. coli L-threonine dehydroge- due was changed to arginine, asparagine, and alanine nase confirm the importance of this residue in cataly- by site-directed mutagenesis in order to probe its role. sis and suggest that His-90 is an active-site residue All three purified, homogeneous mutants, like wild- which modulates the substrate specificity of L-threo- type enzyme, contained one Zn 2/ atom/subunit and ex- nine dehydrogenase.  1998 Academic Press hibited a sequential catalytic mechanism; the k cat Key Words: site-directed mutagenesis; protein isola- value for each, however, was reduced Ç10-fold. The K m tion and purification; steady-state kinetics; substrate value for threonine was elevated from 3 mM for wild- specificity; enzyme active site. 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 transi- L-Threonine dehydrogenase (EC 1.1.1.103) catalyzes tion state. Whereas wild-type enzyme catalyzed the ox- the NAD / -dependent oxidation of threonine to form 2- idation of threonine amide (0.75 M) about twice as fast amino-3-ketobutyrate, which is the first step in the ma- as this same concentration of threonine or 0.375 M L- jor route for threonine metabolism in prokaryotes (1, 2-amino-3-hydroxypentanoate, the reaction rate of 2) and eukaryotes (3). Escherichia coli TDH 4 is a homo- mutant H90A with 0.75 M threonine amide or threo- tetrameric enzyme (M r 149,000) which contains one nine methyl ester was 33- to 35-fold higher than with Zn 2/ atom/subunit (4, 5) and is a member of the me- this level of threonine. Similarly, mutant H90N used dium-chain, zinc-containing alcohol/polyol dehydroge- 0.75 M threonine methyl ester or threonine amide as nase family (6). A progressive alignment of members substrate 9- to 13-fold better than it used this concen- of this group reveals that the amino acid sequence of tration of threonine. Mutants H90A and H90N were E. coli TDH is 26–27% homologous with horse LADH E, human liver ADH, or yeast ADH1 as well as with 1 This research was supported by Grant MCB-9204829 from the human or sheep liver SDH (7). E. coli TDH shows no National Science Foundation. A.R.J. was a predoctoral trainee of similarity with the partial amino acid sequence so far the National Institutes of Health, Research Service Award S-T32- obtained for mammalian (porcine) TDH (8). E. coli GM07767 to the University of Michigan, and was a fellow in the Protein Structure and Design Program of the University of Michigan. 2 Current address: Department of Biochemistry, Parke – Davis 4 Abbreviations used: TDH, L-threonine dehydrogenase; ADH, al- Pharmaceutical Research Division, Warner – Lambert Company, Ann Arbor, MI 48105. cohol dehydrogenase; LADH, horse liver ADH isozyme E; SDH, sorbi- tol (glucitol) dehydrogenase; MNBS, methyl p-nitrobenzenesulfo- 3 To whom correspondence and reprint requests should be ad- dressed. Fax: (313) 763-4581. nate; 2-ME, 2-mercaptoethanol. 8 0003-9861/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.