Park S H, Harris B G, Cook P F
Department of Biochemistry, Texas College of Osteopathic Medicine, Fort Worth.
Biochemistry. 1989 Jul 25;28(15):6334-40. doi: 10.1021/bi00441a027.
Substrate activation of the rate of the NAD-malic enzyme reaction by malate is obtained in the presence but not in the absence of oxalate. The substrate activation is a result of competition between malate and oxalate for the E.NADH complex, with malate binding to the form of the complex unprotonated at an enzyme group with a pK of 4.9 and oxalate binding preferentially to the protonated form. The off-rate for NADH from the E.NADH complex is completely rate limiting when the group with a pK of 4.9 is protonated but is only one of several rate-limiting steps when it is unprotonated [Kiick, D.M., Harris, B.G., & Cook, P.F. (1986) Biochemistry 25, 227]. The competition by malate with oxalate thus results in an overall increase in the off-rate for NADH as a result of binding to the unprotonated form of E.NADH. Consistent with the proposed mechanism, the deuterium isotope effect on V for the nonsubstrate-activating malate concentration range decreases from 1.6 in the absence of oxalate to 1.3 in the presence of a concentration of oxalate equal to its Kii. The rate equation for the oxalate-induced substrate activation by malate is derived and presented in the Appendix. Data are discussed in terms of the overall mechanism of the NAD-malic enzyme.
在有草酸盐存在而不是没有草酸盐存在的情况下，苹果酸可实现对NAD - 苹果酸酶反应速率的底物激活。底物激活是苹果酸和草酸盐竞争E.NADH复合物的结果，苹果酸与复合物的一种形式结合，该形式在pK为4.9的酶基团上未质子化，而草酸盐优先与质子化形式结合。当pK为4.9的基团质子化时，NADH从E.NADH复合物的解离速率完全是限速的，但当它未质子化时，只是几个限速步骤之一[基克，D.M.，哈里斯，B.G.，& 库克，P.F.（1986年）《生物化学》25，227]。因此，苹果酸与草酸盐的竞争导致NADH解离速率总体增加，这是由于它与E.NADH的未质子化形式结合。与所提出的机制一致，在非底物激活的苹果酸浓度范围内，氘同位素对V的影响从没有草酸盐时的1.6降至存在等于其Kii浓度的草酸盐时的1.3。草酸盐诱导苹果酸底物激活的速率方程已推导并列于附录中。根据NAD - 苹果酸酶的整体机制对数据进行了讨论。
