Wright S K, Zhao F J, Rardin J, Milbrandt J, Helton M, Furumo N C
Department of Chemistry, Eastern Illinois University, Charleston 61920, USA.
Arch Biochem Biophys. 1995 Aug 20;321(2):289-96. doi: 10.1006/abbi.1995.1397.
Kinetic studies and chemical modification studies using diethylpyrocarbonate and iodoacetate were performed on malate dehydrogenase isolated from Escherichia coli. Product inhibition experiments indicate that this enzyme follows an ordered Bi Bi kinetic mechanism, similar to other dehydrogenases, while log V/K profiles reveal that one ionizing group with a pKa between 7.8 and 8.7 acts as a general acid/general base in the catalytic mechanism. Log V profiles indicate that malate binds to the correctly protonated form of the enzyme while binding of OAA is pH-independent. Chemical modification experiments implicate an activate site histidine residue is essential for catalytical activity. A primary kinetic isotope effect of 1.44 +/- 0.14 on V/K using malate-2-d at pH 9.0 was measured while no isotope effect was observed on Vmax which is, again, similar to other dehydrogenases. This implies that proton abstraction is partially rate determining under nonsaturating conditions. Within experimental error, small isotope effects were observed on V and V/K (1.219 +/- 0.188 and 1.078 +/- 0.064, respectively) when NADD was utilized indicating that release of cofactor may be rate limiting.
对从大肠杆菌中分离出的苹果酸脱氢酶进行了动力学研究以及使用焦碳酸二乙酯和碘乙酸盐的化学修饰研究。产物抑制实验表明，该酶遵循有序的双底物双产物动力学机制，这与其他脱氢酶类似，而对数V/K曲线显示，一个pKa在7.8至8.7之间的电离基团在催化机制中充当广义酸/广义碱。对数V曲线表明，苹果酸与酶的正确质子化形式结合，而草酰乙酸的结合与pH无关。化学修饰实验表明，一个活性位点组氨酸残基对催化活性至关重要。在pH 9.0条件下，使用苹果酸-2-d测得V/K的一级动力学同位素效应为1.44±0.14，而在Vmax上未观察到同位素效应，这同样与其他脱氢酶相似。这意味着在非饱和条件下，质子抽取部分决定反应速率。在实验误差范围内，当使用NADD时，在V和V/K上观察到较小的同位素效应（分别为1.219±0.188和1.078±0.064），表明辅因子的释放可能是限速步骤。
