Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis.
Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis.
复制标题
压力对酵母醇脱氢酶氘同位素效应的影响：催化机械模型的证据。
作者：
Northrop,DB;Cho,YK
中等压力加速了酵母醇脱氢酶催化的氢化物转移，表明活化的负体积较大[Cho and Northrop（1999）Biochemistry 38，7470−7475]。压力对正常与二氘代苄醇氧化的影响的比较在固有同位素效应中产生双相减少;因此，氘化物转移过渡态的活化体积必须更负，为10.4 mL/mol。这一发现似乎与先前针对该脱氢酶提出的氢隧穿一致[Cha，Y.，默里角，澳-地J.，和Klinman，J.P.（1989）Science 243，1325 - 1330]。然而，一个整体的初步数据拟合表明，整个同位素效应产生的过渡态现象，不像正常的同位素效应，这是由不同的振动频率在反应物状态，隧道同位素效应，这是由两种状态的混合物。假设这种现象是隧道效应，同位素数据与贝尔隧道效应校正因子QH = 12和虚频率νH ε = 1220 cm-1相一致，这是第一个根据实验酶数据计算出的结果。这种过大的校正因子和同位素活化体积的巨大差异，加上外推压力下的低同位素效应，挑战了物理有机化学和过渡态理论在酶催化中的传统应用。相反，他们认为，除了过渡态稳定或隧道效应之外，还有其他因素导致了速率加速，这是酶促过渡态所特有的，在非酶促反应中不会发生。对耦合原子运动的振动模型和蛋白质结构域运动的波动酶模型提出了可能的解释。
Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999)Biochemistry 38, 7470−7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol. This finding appears consistent with hydrogen tunneling previously proposed for this dehydrogenase [Cha, Y., Murray, C. J., and Klinman, J. P. (1989)Science 243, 1325−1330]. However, a global fit of the primary data shows that the entire isotope effect arises from a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling isotope effects, which arise from a mixture of both states. Assuming the phenomenon is tunneling, the isotopic data are consistent with a Bell tunneling correction factor ofQH= 12 and an imaginary frequency of νH‡= 1220 cm-1, the first so calculated from experimental enzymatic data. This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapolated pressures, challenge traditional applications of physical organic chemistry and transition-state theory to enzymatic catalysis. They suggest instead that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic transition state that does not occur in nonenzymatic reactions. Arguments for the vibrational model of coupled atomic motions and the fluctuating enzyme model of protein domain motion are put forward as possible interpretations.