The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis
The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis
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DOI：
10.1021/bi952138d
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发表时间：
1996-04-23
期刊：
影响因子：
2.9
通讯作者：
Kirsch, JF
中科院分区：
文献类型：
--
作者：
Goldberg, JM;Kirsch, JF
研究了大肠杆菌野生型天冬氨酸转氨酶（AATase）和将Tyr-225转化为Phe的突变体AATase （Y225F）催化的转氨酶作用机制。野生型AATase在过量的L-Asp和草酸盐存在下的吸收光谱以在330 nm附近吸收的物种为主。野生型AATase在pH 8.9和7.5条件下对k(cat)/ k - m （Asp）的初生c - α H-2-Asp动力学同位素效应（KIEs）约为2，k（cat）上的KIEs分别为1.9 （pH 8.9）和1.4 （pH 7.5）。由野生型AATase和Y225F催化的L-Asp反应中，k（cat）上的溶剂重氢KIEs （SKIEs）在pH/pD最大值接近2时，k(cat)/ k - m （Asp）上的SKIE由1.3增加到2.3。C-4′(S)- h -2-吡哆胺5′-磷酸在a-酮酸与两种酶反应时的KIE值接近一致。在pH为9时，野生型AATase的黏度对k(cat)/ k - m （Asp）和k（cat）的影响分别为0.10和0.31，说明该反应部分受扩散限制。Y225F的粘度对k(cat)/ k - m （Asp）和k（cat）的影响分别降至-0.02和0.06，表明突变体催化的反应几乎完全受化学限制。野生型AATase构建了l - asp -草酸酯半反应的自由能谱图，c - α H提取、氯胺酮水解和草酸酯解离都部分决定速率。氯胺酮水解是y225f催化反应的唯一速率决定步骤。
The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AATase in which Tyr-225 is converted to Phe (Y225F) was investigated. The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm. The primary C-alpha H-2-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AATase at pH 8.9 and 7.5 on k(cat)/K-M(Asp) are approximately 2, and the KIEs on k(cat) are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C-alpha H-2-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values, The solvent deuterium KIEs (SKIEs) on k(cat) for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximate to 2, and the SKIE on k(cat)/K-M(Asp) is increased from 1.3 to 2.3 by the mutation. The C-4' (S)-H-2-pyridoxamine 5'-phosphate KIE values on reactions of a-ketoacids with both enzymes are near unity. The viscosity effects on k(cat)/K-M(Asp) and k(cat) for wild-type AATase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited. The viscosity effects on k(cat)/K-M(Asp) and k(cat) for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited. A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AATase, C-alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are all partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F-catalyzed reaction.