Mallick S, Harris B G, Cook P F
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
J Biol Chem. 1991 Feb 15;266(5):2732-8.
Initial velocity studies in the absence and presence of product and dead-end inhibitors suggest a steady-state random mechanism for malic enzyme in the direction of reductive carboxylation of pyruvate. For this quadreactant enzymatic reaction (Mn2+ is a pseudoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained at saturating concentrations while one reactant was varied at several fixed concentrations of the other. Data from the resulting reciprocal plots, analyzed in terms of a bireactant mechanism, are consistent with a sequential mechanism with an obligatory order of addition of metal prior to pyruvate. NAD is competitive against NADH whether pyruvate and CO2 are maintained at low or high concentrations, whereas it is noncompetitive against pyruvate and CO2. Thio-NADH, alpha-ketobutyrate, and nitrite were used as dead-end analogs of NADH, pyruvate, and CO2, respectively. Thio-NADH is competitive against NADH, whereas it is noncompetitive against pyruvate and CO2, in accordance with a random mechanism. alpha-Ketobutyrate and nitrite gave noncompetitive inhibition against all substrates. The noncompetitive patterns observed for alpha-ketobutyrate versus pyruvate and nitrite versus CO2 suggest binding of the inhibitor to both the E.Mn.NADH and E.Mn.NAD complexes. Primary deuterium isotope effects are equal on all kinetic parameters, in agreement with the random mechanism, and suggest equal off-rates for NAD from E.Mn.NAD as well as pyruvate and NADH from E.Mn.NADH.pyruvate. Data are consistent with an overall symmetry in the malic enzyme reaction in the two reaction directions with a requirement for metal bound prior to pyruvate and malate.
在不存在和存在产物及终产物抑制剂的情况下进行的初始速度研究表明，苹果酸酶在丙酮酸还原羧化方向上的机制为稳态随机机制。对于这个四反应物酶促反应（Mn2+为假反应物），在两种底物保持饱和浓度而一种反应物在另一种反应物的几个固定浓度下变化的条件下获得了初始速度模式。根据双反应物机制分析所得双倒数图的数据，与丙酮酸之前必须先添加金属的顺序机制一致。无论丙酮酸和CO2保持在低浓度还是高浓度，NAD对NADH均具有竞争性，而对丙酮酸和CO2则无竞争性。硫代-NADH、α-酮丁酸和亚硝酸盐分别用作NADH、丙酮酸和CO2的终产物类似物。根据随机机制，硫代-NADH对NADH具有竞争性，而对丙酮酸和CO2无竞争性。α-酮丁酸和亚硝酸盐对所有底物均产生非竞争性抑制。α-酮丁酸对丙酮酸以及亚硝酸盐对CO2观察到的非竞争性模式表明抑制剂与E.Mn.NADH和E.Mn.NAD复合物均结合。初级氘同位素效应在所有动力学参数上均相等，这与随机机制一致，并表明E.Mn.NAD中的NAD以及E.Mn.NADH.pyruvate中的丙酮酸和NADH的解离速率相等。数据与苹果酸酶反应在两个反应方向上的整体对称性一致，即丙酮酸和苹果酸之前需要结合金属。
