Sekimoto T, Matsuyama T, Fukui T, Tanizawa K
Institute of Scientific and Industrial Research, Osaka University, Japan.
J Biol Chem. 1993 Dec 25;268(36):27039-45.
To elucidate the functional role of the lysyl residue highly conserved in NAD(P)(+)-dependent amino acid dehydrogenases, Lys-80 of leucine dehydrogenase from Bacillus stearothermophilus has been mutated into Ala, Arg, or Gln. All of the mutant enzymes had markedly reduced activities in the oxidative deamination, whereas the Michaelis constants for substrate and coenzyme did not change significantly upon the mutation, except for a 10-30-fold increase in Km values for alpha-keto-iso-caproate in the Ala and Gln mutants. The pH profiles of kinetic parameters of the mutants considerably differed from those of the wild type, in which two ionizable groups with pKa values of 8.9 and 10.7 must be unprotonated for catalysis and protonated for substrate binding, respectively. Combined with the analyses of solvent isotope effect and inhibition by substrate analogs, these results unequivocally show that the epsilon-amino group of Lys-80 participates in catalysis as a general base, assisting the nucleophilic attack of a water molecule to the substrate alpha-carbon atom. Furthermore, the Ala mutant was markedly stimulated by primary amines depending on the pKa and molecular volume, suggesting that in the Ala mutant the added amines can partially replace the general base function of Lys-80 in the wild type enzyme.
为阐明在依赖NAD(P)(+)的氨基酸脱氢酶中高度保守的赖氨酰残基的功能作用，嗜热脂肪芽孢杆菌亮氨酸脱氢酶的第80位赖氨酸已被突变为丙氨酸、精氨酸或谷氨酰胺。所有突变酶的氧化脱氨活性均显著降低，而底物和辅酶的米氏常数在突变后没有明显变化，不过丙氨酸和谷氨酰胺突变体中α-酮异己酸的Km值增加了10 - 30倍。突变体动力学参数的pH曲线与野生型有很大不同，野生型中两个pKa值分别为8.9和10.7的可电离基团，催化时必须去质子化，底物结合时必须质子化。结合溶剂同位素效应分析和底物类似物抑制实验，这些结果明确表明，第80位赖氨酸的ε-氨基作为一般碱参与催化，协助水分子对底物α-碳原子进行亲核攻击。此外，丙氨酸突变体受到伯胺的显著刺激，这取决于pKa和分子体积，表明在丙氨酸突变体中，添加的胺可以部分替代野生型酶中赖氨酸80的一般碱功能。
