Karsten W E, Gavva S R, Park S H, Cook P F
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107.
Biochemistry. 1995 Mar 14;34(10):3253-60. doi: 10.1021/bi00010a015.
The mechanism of the oxidative decarboxylation reaction catalyzed by the NAD-malic enzyme from Ascaris suum has been examined with several different divalent metal ion activators and dinucleotide substrates. Primary deuterium and tritium isotope effects have been obtained and, in combination with the partitioning ratios of the oxalacetate intermediate to malate and pyruvate, have been used to calculate commitment factors, intrinsic deuterium isotope effects on the hydride transfer step, and intrinsic 13C isotope effects for the decarboxylation step. A survey of malate analogs has been undertaken to define the geometry of the active site and to identify functional groups on malate important for substrate binding. With NAD as dinucleotide substrate, a direct correlation between the size of the divalent metal ion activator and the intrinsic deuterium isotope effect is observed. An isotope effect significantly greater than the semiclassical limit is seen when Cd2+ is the metal ion activator, indicating a substantial tunneling contribution. The primary intrinsic 13C isotope effect on the decarboxylation step increases over the series Mg2+ < Mn2+ < Cd2+, which is in contrast to the equal isotope effects measured for these metal ions for the nonenzymatic decarboxylation of oxalacetate [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582]. With Mn2+ or Cd2+ as the divalent metal ion activator, the data support a stepwise mechanism for the enzymatic oxidative decarboxylation with NAD as the dinucleotide substrate, but a change to a concerted mechanism is indicated with more redox-positive dinucleotide substrates as suggested previously with Mg2+ as activator [Karsten, W. E., & Cook, P. F. (1994) Biochemistry 33, 2096].(ABSTRACT TRUNCATED AT 250 WORDS)
利用几种不同的二价金属离子激活剂和二核苷酸底物，对来自猪蛔虫的NAD - 苹果酸酶催化的氧化脱羧反应机制进行了研究。已获得一级氘和氚同位素效应，并结合草酰乙酸中间体向苹果酸和丙酮酸的分配比，用于计算反应决定系数、氢化物转移步骤的固有氘同位素效应以及脱羧步骤的固有13C同位素效应。已对苹果酸类似物进行了研究，以确定活性位点的几何结构，并识别苹果酸上对底物结合重要的官能团。以NAD作为二核苷酸底物时，观察到二价金属离子激活剂的大小与固有氘同位素效应之间存在直接相关性。当Cd2 +作为金属离子激活剂时，同位素效应显著大于半经典极限，表明有大量的隧道效应贡献。脱羧步骤的一级固有13C同位素效应在Mg2 + < Mn2 + < Cd2 +系列中增加，这与这些金属离子对草酰乙酸非酶促脱羧所测得的相同同位素效应形成对比[Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582]。以Mn2 +或Cd2 +作为二价金属离子激活剂时，数据支持以NAD作为二核苷酸底物的酶促氧化脱羧的分步机制，但如先前以Mg2 +作为激活剂时所表明的那样，随着二核苷酸底物的氧化还原电位升高，表明转变为协同机制[Karsten, W. E., & Cook, P. F. (1994) Biochemistry 33, 2096]。（摘要截短于250字）
