Alvarez J A, Gelpí J L, Johnsen K, Bernard N, Delcour J, Clarke A R, Holbrook J J, Cortés A
Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Spain.
Eur J Biochem. 1997 Feb 15;244(1):203-12. doi: 10.1111/j.1432-1033.1997.00203.x.
The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD+ plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD+-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH-->NAD+ direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast. The release of NAD+ at pH 8.0 is 200-times faster than Kcat (NADH-->NAD+) whereas the release of NADH is only 5-times faster than Kcat (NAD+-->NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E-NADH complex) and < or = 6.4 (in the E-NAD+ complex) which may be the proton donor and acceptor for the chemical reaction. At pH 6.5 the mechanism changes in the NADH-->NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+. In addition the inhibition by excess oxo-4-methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids.
已通过初速度、产物抑制和终产物抑制技术在pH 8.0条件下研究了D-2-羟基-4-甲基戊酸脱氢酶的稳态动力学。该机制在NAD⁺加D-2-羟基-4-甲基戊酸方向上是快速平衡有序的，而在另一个方向上是稳态有序的。在这两种情况下，辅酶都是首先添加的底物，并且E-NADH-D-2-羟基-4-甲基戊酸和E-NAD⁺-2-氧代-4-甲基戊酸都会产生无效复合物，从而导致过量底物抑制。稳态测量表明，在pH 8.0时两个方向上的限速步骤都在酶-辅酶-底物三元复合物的形成与反应的第一个产物的释放之间。瞬态动力学与初级动力学氘同位素效应相结合表明，在NADH→NAD⁺方向上，E-NADH-氧代酸复合物存在缓慢的限速重排，而氢化物转移非常快。在pH 8.0时NAD⁺的释放速度比Kcat（NADH→NAD⁺）快200倍，而NADH的释放速度仅比Kcat（NAD⁺→NADH）快5倍。NADH结合的pH依赖性取决于两个pKa约为5.9的可电离残基的存在。动力学参数的pH依赖性由第三个pKa值为7.2（在E-NADH复合物中）和≤6.4（在E-NAD⁺复合物中）的可电离残基解释，该残基可能是化学反应的质子供体和受体。在pH 6.5时，NADH→NAD⁺方向上的机制发生变化，部分受化学步骤限制，测得的初级动力学同位素效应为5.7，部分受NAD⁺的解离速度仅略快限制。此外，过量的氧代-4-甲基戊酸的抑制作用更明显。该机制意味着去除由控制辅酶亲和力的两个基团产生的正电荷，既可以提高pH 6.5时的催化速率，又可以减少过量底物抑制，从而提供一种更适合大量合成D-2-羟基酸的酶。
