Park S H, Kiick D M, Harris B G, Cook P F
Biochemistry. 1984 Nov 6;23(23):5446-53. doi: 10.1021/bi00318a011.
Measurement of the initial rate of the malic enzyme reaction varying the concentration of NAD at several different fixed levels of Mg2+ (0.25-1.0 mM) and a single malate concentration gave a pattern which intersects to the left of the ordinate. Repetition of this initial velocity pattern at several additional malate concentrations and treatment in terms of a terreactant mechanism suggests an ordered mechanism in which NAD adds prior to Mg2+ which must add prior to malate. On the other hand, when a broader concentration range of Mg2+ (0.25-50 mM) is used, data are consistent with a random mechanism in which Mg2+ must add prior to malate. By use of product inhibition studies, pyruvate is competitive vs. malate and noncompetitive vs. NAD, while NADH is competitive vs. NAD and noncompetitive vs. malate. These results are consistent with the random addition of substrates and further suggest rapid equilibrium random release of products. Tartronate, a dead-end analogue of malate, is competitive vs. malate and noncompetitive vs. NAD. Thio-NAD is a slow substrate which is used at 2.4% the maximum rate of NAD. When used as a dead-end analogue of NAD, thio-NAD is competitive vs. NAD and gives a complex inhibition pattern vs. malate in which competitive inhibition is apparent at low concentrations of malate (less than 12.5 mM), and this changes to uncompetitive inhibition at high concentrations of malate (greater than 12.5 mM). These data are consistent with a steady-state random mechanism in the direction of oxidative decarboxylation in which Mg2+ adds in rapid equilibrium prior to malate.(ABSTRACT TRUNCATED AT 250 WORDS)
在几种不同固定水平的Mg2+（0.25 - 1.0 mM）和单一苹果酸浓度下，通过改变NAD浓度来测量苹果酸酶反应的初始速率，得到的模式在纵坐标左侧相交。在几个额外的苹果酸浓度下重复这种初始速度模式，并根据三反应物机制进行处理，表明是一种有序机制，其中NAD在Mg2+之前添加，而Mg2+必须在苹果酸之前添加。另一方面，当使用更宽的Mg2+浓度范围（0.25 - 50 mM）时，数据与一种随机机制一致，即Mg2+必须在苹果酸之前添加。通过产物抑制研究，丙酮酸对苹果酸具有竞争性，对NAD无竞争性，而NADH对NAD具有竞争性，对苹果酸无竞争性。这些结果与底物的随机添加一致，并进一步表明产物的快速平衡随机释放。丙醇二酸是苹果酸的一种终产物类似物，对苹果酸具有竞争性，对NAD无竞争性。硫代-NAD是一种慢底物，其使用速率为NAD最大速率的2.4%。当用作NAD的终产物类似物时，硫代-NAD对NAD具有竞争性，对苹果酸产生复杂的抑制模式，其中在低浓度苹果酸（小于12.5 mM）时竞争性抑制明显，而在高浓度苹果酸（大于12.5 mM）时变为非竞争性抑制。这些数据与氧化脱羧方向上的稳态随机机制一致，其中Mg2+在苹果酸之前以快速平衡的方式添加。（摘要截短至250字）
