Edens W A, Urbauer J L, Cleland W W
Institute for Enzyme Research, University of Wisconsin, Madison 53705, USA.
Biochemistry. 1997 Feb 4;36(5):1141-7. doi: 10.1021/bi962128s.
Carbon-13 isotope effects have been determined for all four carbons of L-malate as a substrate for chicken liver malic enzyme, using either NADP or acetylpyridine-NADP as the other substrate. The effect of deuteration at C2 of malate was then used to tell whether the chemical mechanism of this oxidative decarboxylation was stepwise, with oxaloacetate as an intermediate, or concerted. With NADP, the 13C isotope effects at C3 and C4 both decrease with deuteration of malate, showing a stepwise mechanism, as previously determined [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114]. With acetylpyridine-NADP, however, the 13C isotope effects at both C3 and C4 increase with deuteration of malate. While the increase at C4 could be explained by a secondary 13C isotope effect on hydride transfer, the increase at C3 proves that the chemical mechanism has changed to a concerted one, presumably because hydride transfer is more rate-limiting and the overall equilibrium constant is more favorable by 2 orders of magnitude. The transition state for this concerted reaction is asynchronous, however, with an intrinsic deuterium isotope effect of approximately 5 and a 13C isotope effect of only 1.010-1.015. Equilibrium 13C isotope effects for conversion of carbons 2, 3, and 4 of malate to pyruvate or CO2 are 1.010, 1.011, and 0.988, respectively. Measured 13C isotope effects at C2 of malate are slightly inverse, but no explanation for this is obvious. With NADP, deuterium isotope effects at C3 of 1.17 and 1.08 for di- and monodeuteration and an increase in the 13C isotope effect at C4 upon dideuteration at C3 are consistent with a stepwise mechanism with the deuterium isotope effect at C3 being only on the decarboxylation step. Smaller deuterium isotope effects of 1.03-1.04 from dideuteration at C3 with acetylpyridine-NADP are consistent with a concerted but asynchronous mechanism where C-C cleavage is not far advanced in the transition state.
以L-苹果酸为底物、NADP或乙酰吡啶-NADP为另一底物，测定了鸡肝苹果酸酶作用下L-苹果酸所有四个碳原子的碳-13同位素效应。随后利用苹果酸C2位的氘代效应来判断这种氧化脱羧反应的化学机制是分步的（以草酰乙酸为中间体）还是协同的。对于NADP，随着苹果酸的氘代，C3和C4位的13C同位素效应均降低，表明是分步机制，如先前所确定的[赫姆斯，J.D.，罗斯克，C.A.，奥利里，M.H.，&克莱兰，W.W.（1982年）《生物化学》21，5106 - 5114]。然而，对于乙酰吡啶-NADP，随着苹果酸的氘代，C3和C4位的13C同位素效应均增加。虽然C4位的增加可以用氢化物转移上的二级13C同位素效应来解释，但C3位的增加证明化学机制已转变为协同机制，大概是因为氢化物转移更具速率限制性，且总平衡常数更有利两个数量级。然而，这种协同反应的过渡态是异步的，其内在氘同位素效应约为5，13C同位素效应仅为1.010 - 1.015。苹果酸的2、3和4位碳原子转化为丙酮酸或二氧化碳的平衡13C同位素效应分别为1.010、1.011和0.988。在苹果酸C2位测得的13C同位素效应略有反向，但对此并无明显解释。对于NADP，二氘代和单氘代时C3位的氘同位素效应分别为1.17和1.08，以及在C3位进行二氘代时C4位的13C同位素效应增加，这与分步机制一致，其中C3位的氘同位素效应仅在脱羧步骤上。用乙酰吡啶-NADP在C3位进行二氘代时较小的氘同位素效应为1.03 - 1.04，这与协同但异步的机制一致，即C - C键断裂在过渡态中进展不大。
