Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects.
Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects.
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通过同位素效应测定天然和替代二核苷酸的人苹果酸酶的机制。
DOI：
10.1006/abbi.2001.2598
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Cleland,WW
中科院分区：
文献类型：
--
作者：
Rishavy,MA;Yang,Z;Tong,L;Cleland,WW
用稳态动力学、氘同位素效应和13 C同位素效应研究了人苹果酸酶与生理二核苷酸辅因子和几种替代辅因子的关系。NAD的log V vs pH曲线显示两个pKa值太接近而无法单独测定，但平均值为7.33。NAD的log V/K vs pH曲线显示两个pKa值，分别为7.4和5.6。氘和13 C同位素效应表明，人苹果酸酶的机制是逐步与NAD和NAD的，但超共轭的过渡态的氢化物转移是可检测的，只有前者。对于thioNAD和APAD，同位素效应不能清楚地表明该机制是逐步的还是协同的。通过测量在NADH和人苹果酸酶存在下草酰乙酸的分配比（脱羧为丙酮酸/还原为苹果酸= 2.33），计算出脱羧的固有13 C同位素效应为1.0485。同位素效应和分区数据表明，草酰乙酸脱羧的能量势垒是不高的相对障碍，减少草酰乙酸与鸡肝酶。
Human malic enzyme was studied by steady state kinetics, deuterium isotope effects, and13C isotope effects with both the physiological dinucleotide cofactor and several alternate cofactors. The log V vs pH profile with NAD revealed two pKavalues too close to be separately determined, but with an average value of 7.33. The log V/K vs pH profile with NAD revealed two pKavalues at 7.4 and 5.6. Deuterium and13C isotope effects indicate that the mechanism of human malic enzyme is stepwise with both NAD and ϵNAD, but that hyperconjugation in the transition state for hydride transfer is detectable only with the former. With thioNAD and APAD, the isotope effects do not clearly indicate whether the mechanism is stepwise or concerted. The intrinsic13C isotope effect for decarboxylation was calculated to be 1.0485 by measurement of the partition ratio of oxaloacetate in the presence of NADH and human malic enzyme (decarboxylation to pyruvate/reduction to malate = 2.33). The isotope effect and partitioning data suggest that the energy barrier for decarboxylation of oxaloacetate is not as high relative to the barrier for reduction of oxaloacetate as with the chicken liver enzyme.