Rajapaksa R, Abu-Soud H, Raushel F M, Harris B G, Cook P F
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Biochemistry. 1993 Mar 2;32(8):1928-34. doi: 10.1021/bi00059a007.
Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting a slow release of NADH [Kiick, D. M., Harris, B. G., & Cook, P. F. (1986) Biochemistry 25, 227-236]. The latter, however, requires a burst in the pre-steady-state time course, and thus the previous data have been reinterpreted in view of the observed lag. Preincubation with NAD and/or Mg increases the lag rate, with the latter having the greater effect, while preincubation with Mg and malate (or a malate analog) eliminates the lag. Data suggest a slow isomerization of E:NAD that is increased by addition of malate prior to NAD in the presence of Mg. The lag is also eliminated at low pH as a result of the overall rate being limited by the isomerization; that is, the isomerization is pH-dependent. Fumarate, an activator of the NAD-malic enzyme, when preincubated with enzyme also eliminates the lag, suggesting that the activator preferentially binds the isomerized form of the enzyme or increases the isomerization rate, or both. Stopped-flow data are corroborated by circular dichroism experiments. The unliganded enzyme is approximately 50% alpha-helix on the basis of secondary structural analysis. Binding of NAD and Mg exhibits a substantial change, with a further change observed upon binding the malate analog tartronate.
在NAD - 苹果酸酶反应的预稳态时间尺度上进行的停流实验表明，在达到稳态之前存在一个滞后阶段。以前通过同位素效应研究得到的结果显示，在低pH值下氘同位素对Vmax的效应降低到1，这一结果被解释为暗示NADH的缓慢释放[基克，D. M.，哈里斯，B. G.，& 库克，P. F.（1986年）《生物化学》25卷，227 - 236页]。然而，后者需要在预稳态时间进程中有一个爆发阶段，因此根据观察到的滞后现象对以前的数据进行了重新解释。用NAD和/或镁进行预孵育会增加滞后速率，其中镁的影响更大，而用镁和苹果酸（或苹果酸类似物）进行预孵育则消除了滞后现象。数据表明，在镁存在的情况下，先加入苹果酸再加入NAD会增加E:NAD的缓慢异构化。由于整体速率受异构化限制，在低pH值下滞后现象也会消除；也就是说，异构化是pH依赖性的。富马酸是NAD - 苹果酸酶的激活剂，当与酶预孵育时也会消除滞后现象，这表明激活剂优先结合酶的异构化形式或增加异构化速率，或两者兼而有之。圆二色性实验证实了停流数据。根据二级结构分析，未结合配体的酶大约50%为α - 螺旋结构。NAD和镁的结合表现出显著变化，在结合苹果酸类似物酒石酸时还观察到进一步的变化。
