PRE-STEADY-STATE KINETICS OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE CATALYZED-REACTIONS AND THERMODYNAMIC ASPECTS OF ITS SUBSTRATE-SPECIFICITY
PRE-STEADY-STATE KINETICS OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE CATALYZED-REACTIONS AND THERMODYNAMIC ASPECTS OF ITS SUBSTRATE-SPECIFICITY
复制标题
DOI：
10.1021/bi00475a010
复制
发表时间：
1990-06-12
期刊：
影响因子：
2.9
通讯作者：
KAGAMIYAMA, H
中科院分区：
文献类型：
--
作者：
KURAMITSU, S;HIROMI, K;KAGAMIYAMA, H
在停流光谱仪中监测333或358 nm处的吸光度变化，跟踪四个半转氨化反应[大肠杆菌天冬氨酸氨基转移酶(Aspat)与天冬氨酸或谷氨酸的吡哆醇形式，以及该酶与草乙酸酯或2-氧戊二酸的吡哆胺形式]。所有情况下的反应进程曲线均符合单相指数过程。这些反应的动力学分析表明，每个半反应由以下三个过程组成：(1)氨基酸底物与酶的吡哆醛的快速结合；(2)相应的酮酸与酶的吡哆胺的快速结合；(3)两个复合体之间的速度决定的相互转化。半转氨化反应和全转氨化反应的平衡常数以及稳态动力学常数(Km和kcat)与基于上述机理使用稳态前动力学参数的预测值符合得很好。在与氚氨基酸的反应中观察到的显著的初级动力学同位素效应表明，底物的α-质子的退出是速率决定的。大肠杆菌Aspat的吡哆醇形式与多种氨基酸作为底物发生反应。根据稳态前的动力学参数计算出过渡态和非结合态(非结合酶+自由底物)之间的Gibbs自由能差，表明吉布斯自由能差与含有未带电侧链的氨基酸底物的可及表面积呈线性关系。大肠杆菌酶的底物专一性比猪的同工酶要宽得多，反映了大肠杆菌和哺乳动物AspATs在底物侧链的微环境上的一些细微而明显的差异。
The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two commplexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the .alpha.-proton of the substrates is rate determining. The pyridoxal form of A. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain. The substrate specificity of the E. coli enzyme as much broader than that of pig isoenzymes, reflecting some subtle but distinct difference in microenvironment accommodating the side chain of the substrate between E. coli and mammalian AspATs.