Johnson A R, Dekker E E
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Arch Biochem Biophys. 1998 Mar 1;351(1):8-16. doi: 10.1006/abbi.1997.0501.
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.
大肠杆菌L-苏氨酸脱氢酶是含锌（2+）的醇/多元醇脱氢酶家族的一员。最近发现L-苏氨酸脱氢酶His-90的甲基化会导致完全失活（J.P. Marcus和E.E. Dekker，1995年，《生物化学与生物物理学报》316卷，413 - 420页）。由于His-90在相关脱氢酶中并不保守，通过定点诱变将该残基分别替换为精氨酸、天冬酰胺和丙氨酸，以探究其作用。所有三种纯化的同源突变体，与野生型酶一样，每个亚基含有一个Zn2+原子，并表现出有序催化机制；然而，每个突变体的kcat值大约降低了10倍。苏氨酸的K(m)值从野生型酶的3 mM分别升高到突变体H90R、H90N和H90A的31 mM、328 mM和417 mM。突变体H90A催化反应的活化能增加了6.6千卡/摩尔，这表明在野生型酶中，His-90在过渡态至少形成了一个关键的氢键。野生型酶催化0.75 M苏氨酸酰胺的氧化速度约为相同浓度苏氨酸或0.375 M L-2-氨基-3-羟基戊酸的两倍，而突变体H90A与0.75 M苏氨酸酰胺或苏氨酸甲酯的反应速率比与该浓度苏氨酸的反应速率高33至35倍。同样，突变体H90N以0.75 M苏氨酸甲酯或苏氨酸酰胺作为底物的反应效果比以该浓度苏氨酸作为底物的反应效果好9至13倍。突变体H90A和H90N与0.225 M L-苏氨酸异羟肟酸的反应活性比与0.75 M苏氨酸的反应活性更高，但突变体H90A不氧化0.375 M L-2-氨基-3-羟基戊酸，突变体H90N对该底物的利用效果较差。突变体H90R的最佳底物是苏氨酸甲酯、苏氨酸和苏氨酸酰胺（均在0.75 M浓度下测试）；0.375 M L-2-氨基-3-羟基戊酸是一种较差的底物。这些大肠杆菌L-苏氨酸脱氢酶的首个His-90突变体的分离和特性研究证实了该残基在催化中的重要性，并表明His-90是一个活性位点残基，可调节L-苏氨酸脱氢酶的底物特异性。
