Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase.
Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase.
复制标题
高压对酵母乙醇脱氢酶溶剂同位素效应的影响。
DOI：
10.1016/s0006-3495(00)76412-5
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发表时间：
2000
影响因子：
3.4
通讯作者：
Cho,YK
中科院分区：
文献类型：
--
作者：
Northrop,DB;Cho,YK
压力对酵母醇脱氢酶捕获底物醇的影响是双相的。溶剂同位素效应伴随这两个阶段，并在不同的压力下表现不同。这些差异允许提取1.1的逆本征动力学溶剂同位素效应（即，D2 OV/K=0.9）伴随氢化物转移和2.6的逆平衡溶剂同位素效应（即，D_2OKs =0.4），伴随核苷酸NAD ~+的结合。的动力学效应的值是一致的反应器状态E-NAD+-Zn-OH 2具有分馏因子为0.5的锌结合的水与过渡态质子退出低势垒氢键与分馏因子之间的0.6和0.9。平衡效应的值与伴随NAD+结合的构象变化期间酶蛋白的多个氢的扭转运动的限制是一致的。伴随动力学溶剂同位素效应的催化作用的缺乏意味着这部分质子转移发生在与协同化学机制中的氢化物转移相同的反应步骤中。这种分析的成功表明，未来的测量溶剂同位素效应作为压力的函数，在存在适度的承诺催化，可能会产生精确的估计内在的溶剂同位素效应，在大气压下捕获不完全表示。
The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e.,D2OV/K=0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e.,D2OKs=0.4) accompanying the binding of nucleotide, NAD+. The value of the kinetic effect is consistent with a reactant-state E-NAD+-Zn-OH2having a fractionation factor ofϕ≈0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD+. The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.