Alternative substrates for malic enzyme: oxidative decarboxylation of L-aspartate.
Alternative substrates for malic enzyme: oxidative decarboxylation of L-aspartate.
复制标题
苹果酸酶的替代底物：L-天冬氨酸的氧化脱羧。
作者：
Dali Liu;C. Hwang;P. Cook
来自Ascaris suum的nad -苹果酸酶将利用l -天冬氨酸，(2S,3R)-酒石酸盐和中酒石酸盐作为底物，相对于苹果酸盐，V/K值为10(-4)-10（-5）。对所有这些反应物的2S立体化学都有严格的要求。由于天冬氨酸是苹果酸酶的一种独特的氨基酸反应物，因此确定其氧化脱羧机制的细节是有意义的。NADH出现的初始速率与天冬氨酸的浓度成正比，难以达到饱和。V/K（天冬氨酸）E(t)的pH依赖性在低pH下降低，pK为5.7。与ph无关的V/K（天冬氨酸）E(t)值为3 M(-1) s(-1)，比l -苹果酸低12500倍。在中性pH下，天冬氨酸作为苹果酸竞争性抑制剂的解离常数为60 mM，与l -苹果酸获得的39 s（-1）的V/E(t)相比，l -天冬氨酸的解离常数约为0.18 s（-1）。在5.1 ~ 6.9范围内，氘同位素对V/K（天冬氨酸）的影响与pH无关，平均值为3.3。数据表明，l -天冬氨酸的单阴离子与酶结合，并且负责苹果酸氧化脱羧生成丙酮酸的一般碱、一般酸机制也适用于天冬氨酸氧化脱羧生成亚氨基丙酮酸。此外，以天冬氨酸为底物，氧化步骤似乎主要是速率决定的。
The NAD-malic enzyme from Ascaris suum will utilize L-aspartate, (2S,3R)-tartrate, and meso-tartrate as substrates with V/K values 10(-4)-10(-5) with respect to malate. There is a strict requirement for the 2S stereochemistry for all of these reactants. Since aspartate is unique as an amino acid reactant for malic enzyme, it was informative to determine the details of its mechanism of oxidative decarboxylation. The initial rate of NADH appearance is directly proportional to the concentration of aspartate, and saturation is difficult to achieve. The pH dependence of V/K(aspartate)E(t) shows a decrease at low pH, giving a pK of 5.7. The pH-independent value of V/K(aspartate)E(t) is 3 M(-1) s(-1), 12500-fold lower than that obtained with L-malate. The dissociation constant for aspartate as a competitive inhibitor of malate is 60 mM at neutral pH, allowing an estimate of about 0.18 s(-1) for V/E(t) with L-aspartate compared to a value of 39 s(-1) obtained with L-malate. The deuterium isotope effect on V/K(aspartate) is pH independent over the range 5.1-6.9 with an average value of 3.3. Data suggest that the monoanion of L-aspartate binds to enzyme and that the same general base, general acid mechanism that is responsible for the oxidative decarboxylation of malate to pyruvate applies to the oxidative decarboxylation of aspartate to iminopyruvate. In addition, the oxidation step appears to be largely rate determining with aspartate as the substrate.