PROTONATION MECHANISM AND LOCATION OF RATE-DETERMINING STEPS FOR THE ASCARIS-SUUM NICOTINAMIDE ADENINE-DINUCLEOTIDE MALIC ENZYME REACTION FROM ISOTOPE EFFECTS AND PH STUDIES
PROTONATION MECHANISM AND LOCATION OF RATE-DETERMINING STEPS FOR THE ASCARIS-SUUM NICOTINAMIDE ADENINE-DINUCLEOTIDE MALIC ENZYME REACTION FROM ISOTOPE EFFECTS AND PH STUDIES
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DOI：
10.1021/bi00349a032
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发表时间：
1986-01-14
期刊：
影响因子：
2.9
通讯作者：
COOK, PF
中科院分区：
文献类型：
--
作者：
KIICK, DM;HARRIS, BG;COOK, PF
确定了动力学参数的 pH 依赖性以及以烟酰胺腺嘌呤二核苷酸 (NAD) 和硫代烟酰胺腺嘌呤二核苷酸 (thio-NAD) 作为核苷酸底物的初级氘同位素效应，以获得有关猪蛔虫 NAD-苹果酸酶反应的化学机制和速率决定步骤位置的信息。使用 thio-NAD 作为核苷酸时，最大速度在 pH 4.2 至 9.6 范围内与 pH 值无关，而使用 NAD 时，V 降低至 pK 4.8 以下。两种核苷酸的 V/K 在 pK 低于 5.6 和高于 pK 8.9 时降低。当 pK 低于 4.8 和高于 8.9 时，酒石酸 pKi 和 V/Kmalate 均降低。当 NAD 作为核苷酸时，草酸盐在 pH 7 以上时与苹果酸盐具有竞争性，而在 pH 7 以下则不具有竞争性。草酸盐 Kis 从 pK 4.9 以上的恒定值增加到 pK 6.7 以上的另一个恒定值。草酸盐 Kii 也会增加到 pK 4.9 以上，并且这种抑制作用会被 NADH 增强。在 thio-NAD 存在的情况下，草酸盐的抑制作用与 pH 7 以下的苹果酸盐具有竞争性。对于 thio-NAD，DV 和 D(V/K) 均与 pH 值无关，且等于 1.7。以 NAD 作为核苷酸，DV 在 pK 4.9 以下降至 1.0，而 D(V/KNAD) 和 D(V/Kmalate) 与 pH 无关。 pH 7 以上时，同位素对 V 的影响以及 NAD 和苹果酸的 V/K 值等于 1.45，即 DV 在 pH 7 以上时与 pH 无关的值。根据上述数据，可以得出有关该酶的机制的以下结论。底物仅与酶的正确质子化形式结合。两个酶组对于底物的结合和催化是必需的。 NAD 和苹果酸均以相同的速率从米氏复合体中释放，该速率等于 pH 7 以上时 E-NADH 中 NADH 的释放速率。pH 7 以下时，随着 pH 值的降低，NADH 的释放变得更具速率决定性，直到 pH 4.0 时，它完全限制了反应的总体速率。
The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.