Mechanistic Studies on the Lactate Dehydrogenase Reaction

乳酸脱氢酶通过促进一个质子和两个电子同时直接转移到辅酶NAD+上来催化乳酸的氧化。由于许多其他脱氢酶以类似的方式运作，因此提出的研究可以作为未来解释脱氢酶如何实现化学反应的大幅加速的模型。利用化学动力学和拉曼光谱来确定底物和辅酶与酶结合时化学键的变化，这些已知的底物和辅酶结合的酶的结构变化将是相关的。这些研究将部分涉及合成同位素标记的底物和辅酶，以便拉曼能带可以分配给这些分子在溶液中和与酶结合的分子运动。这些研究还将扩展到甲酸脱氢酶，以对发生的相互作用产生更全面的了解。最近的研究表明，在很大程度上，酶利用非共价相互作用来促进共价键的形成和断裂，并在空间上形成或断裂它们。由于在酶反应过程中，非共价相互作用的能量不容易评估，因此描述各种类型的非共价相互作用对酶催化的贡献是一个困难但重要的目标。根据观察到的由简单催化剂产生的速率效应来使酶反应合理化的可能性是酶化学家多年来的目标。由于这项工作，这可能是NAD依赖性脱氢。

Lactate dehydrogenase catalyzes the oxidation of lactate by facilitating the direct, simultaneous transfer of a proton and two electrons to the coenzyme, NAD+. Because many other dehydrogenases operate in a similar manner, the proposed research serves as a model for future explanations of how dehydrogenases accomplish large accelerations of chemical reactions. Using both chemical kinetics and Raman spectroscopy to determine changes in the chemical bonds of both the substrate and coenzyme upon binding to the enzyme, these changes to the structure, which is known, of the enzyme with substrate and coenzyme bound will be related. These studies will partially involve the synthesis of isotopically labeled substrates and coenzymes so that Raman bands can be assigned to the molecular motions of these molecules both in solution and bound to enzyme. These studies also will be extended to formate dehydrogenase to produce a more general picture of the interactions that occur. Recent work shows that to a large extent enzymes use noncovalent interactions to facilitate the making and breaking of covalent bonds and to make or break them sterically. Because the energies of noncovalent interactions are not easily assessed during an enzyme reaction a description of the contributions to enzyme catalysis provided by various types of noncovalent interactions is a difficult yet important goal, and the possibility of rationalizing enzymic reactions in terms of the observed rate effects produced by simple catalysts has been the goal of enzyme chemists for many years. Because of this work this may be possible for NAD dependent dehydrogenasis.