Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Biochem J. 2009 Dec 23;425(2):455-63. doi: 10.1042/BJ20091441.
The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.
Asn191 和 Asn300 的侧链构成了荧光假单胞菌甘露醇 2-脱氢酶活性部位的一个特征结构基序，该基序在已知的醇脱氢酶中没有先例，类似于丝氨酸蛋白酶的典型氧阴离子结合口袋。我们使用稳态和瞬态动力学研究了不同 pH 值和底物和溶剂中氘同位素取代对酶反应速率的影响，以描绘由两个天冬酰胺残基单独和组合取代为丙氨酸所导致的催化后果。氢化物从甘露醇 C-2 向酶的总体转移速率常数，在野生型酶中分别约为 5 x 102 s-1 和约 1.5 x 103 s-1，在单一位点甘露醇 2-脱氢酶突变体中分别被选择性地减缓了 540-和 2700 倍。这些影响在相应的双突变酶中是相加的，表明两个天冬酰胺残基在催化中独立发挥作用。单一位点突变体中氧阴离子孔的部分破坏导致酶-NAD+-甘露醇复合物中催化酸碱 Lys295 的动力学 pK 值向上移动>或=1.2 pH 单位。甘露醇 2-脱氢酶的氧阴离子孔被认为在三元复合物水平上驱动一个预催化构象平衡，其中底物的反应基团通过与未质子化的 Lys295 侧链的精确对齐（甘露醇氧化）和 Asn191 和 Asn300 的羧酰胺部分对 C=O 键的极化而被“激活”进行化学转化（果糖还原）。在随后的氢化物转移步骤中，两个天冬酰胺残基提供了约 40 kJ/mol 的静电稳定化。
