Effect of pressure on deuterium isotope effects of formate dehydrogenase.
Effect of pressure on deuterium isotope effects of formate dehydrogenase.
复制标题
压力对甲酸脱氢酶的氘同位素效应的影响。
DOI：
10.1021/bi001991w
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Northrop,DB
中科院分区：
文献类型：
--
作者：
Quirk,DJ;Northrop,DB
高压导致酵母甲酸脱氢酶氧化甲酸盐的两相效应，如动力学参数V/K所示，该参数衡量底物捕获。中等压力通过加速氢化物转移来增加捕获量。氢化物转移的过渡态比游离甲酸盐加酶的捕获态体积小，ΔV‡=−为9.7±1.0mL/m ol。高于1.5kbar的压力会降低捕获量，这让人想起与烟酰胺腺嘌呤二核苷酸(NAD+)与酵母酒精脱氢酶结合有关的构象变化的影响[Northrop，D.B.，and Y.K.Cho(2000)BioChemstry39,2406−2412]。碰撞络合物E-NAD+的体积比结合更紧密的反应态络合物E*-NAD+的体积小，ΔV*=+83.4±5.2mL.压力对正常和重甲酸氧化的影响的比较表明，氢化物转移的整个同位素效应2.73±0.20完全来自过渡态现象，就像以前在酵母醇脱氢中观察到的那样。相反，正常的初级同位素效应仅由反应态中不同的零点能量引起，而那些表示氢隧穿的效应则来自反应态和过渡态现象的混合。此外，压力增加了初级本征的氚同位素效应，这与酵母酒精脱氢所观察到的相反。同位素效应的缺乏也与怀疑隧穿的化学反应的经验先例相反，也与酶反应中振动增强隧穿的理论构造相反。因此，这一新的实验设计前所未有地穿透了酶催化的过渡态，揭示了化学动力学以外的现象的存在，并呼吁解释酶是如何超越物理有机化学的。
High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameterV/K, which measures substrate capture. Moderate pressure increases capture by accelerating hydride transfer. The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with ΔV‡= −9.7 ± 1.0 mL/mol. Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD+) to yeast alcohol dehydrogenase [Northrop, D. B., and Y. K. Cho (2000)Biochemistry39, 2406−2412]. The collision complex, E-NAD+, has a smaller volume than the more tightly bound reactant-state complex, E*-NAD+, with ΔV* = +83.4 ± 5.2 mL/mol. A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 ± 0.20, arises solely from transition-state phenomena, as was also observed previously with yeast alcohol dehydrogense. In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena. Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions. Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.