KINETIC & CHEMICAL MECHANISMS OF ENZYMES

本提案的总体目标是了解酶 机制 努力集中在三种酶系统上，包括 苹果酸酶、磷酸果糖激酶和葡萄糖氧化酶。三步化学 已经提出了苹果酸酶的机制，其中苹果酸是第一个 氧化成乙酰乙酸酯，乙酰乙酸酯中间体脱羧， 烯醇丙酮酸，后者然后互变异构为丙酮酸。 最近 用替代的二核苷酸底物获得的证据表明， 有两种可能第一，改革的机制发生了变化， 从两步到一步的苹果酸氧化脱羧为烯醇丙酮酸 协同机制其次，存在次级（13）C同位素效应 在苹果酸盐氧化成乙酸盐中间体的过程中。的 在E：NADH：Mg：Na 2CO 3乙酸盐中分配Na 2CO 3乙酸盐中间体 复杂的，对苹果酸和丙酮酸将被用来探测这一可能 机制变革。这些研究将用protium进行， 氘标记的还原的替代性二核苷酸和Mg（2+）、Mn（2+）和 Cd（2+）。这些研究将采用次级氘同位素进行后续研究。 使用NAD-4-D和L-苹果酸-3，3-t2定义过渡态的影响 用于氢化物转移和脱羧（如果存在）步骤的结构。在 此外，初级氘和氚同位素效应将用于 研究还原羧化反应以及金属的作用 离子。研究将扩展到包括密切相关的异柠檬酸和 δ-磷酸葡糖酸脱氢酶，以确定是否现象 上述苹果酸酶是这类氧化酶所共有的。 脱羧酶初步证据显示 焦磷酸盐中的第二金属离子（除了MgPPi之外） 磷酸果糖激酶（PPi-PFK）反应。 交换惰性金属-PPi配合物 将被用来检验这一假设。磷酰基转移步骤是速率 确定PPi-PFK反应，从而确定初级和次级（18）O 将使用远程标记技术进行效果探测， 过渡态结构ATP-PFK形式的可用性 对F6 P磷酸化的时间进程中的滞后不敏感， 同向协同效应促进了动力学机理的研究 调节和酸碱催化的机制。初始速度 研究将用于确定变构调节剂沿着的作用 反应途径、酸碱作用机理、催化作用、 反应物和效应物上结合基团的最佳质子化状态， 以及酶上的活性和变构位点。E1 cb机制具有 已被提出为其中C-N键断裂是速率决定的酶。 将使用NMR中的氚洗脱来测试所提出的机制。 此外，还对该酶的酸碱催化机理和最适反应条件进行了探讨 将确定结合基团的质子化状态。

The overall objective of this proposal is an understanding of enzyme mechanism. Efforts are concentrated on three enzyme systems including the malic enzyme, phospho-fructokinase, and aspartase. A three step chemical mechanism has been proposed for malic enzyme in which malate is first oxidized to oxalaceate, the oxalaceate intermediate is decarboxylated to enolpyruvate, and the latter is then tautomerized to pyruvate. Recent evidence obtained with alternative dinucleotide substrates suggests one of two possibilities. First, there is a change in the mechanism of the oxidative decarboxylation of malate to enolpyruvate from two steps to a concerted mechanism. Second, a secondary (13)C isotope effect is present during the oxidation of malate to the oxalacetate intermediate. The partitioning of the oxalacetate intermediate in the E:NADH:Mg:oxalacetate complex, toward malate and pyruvate will be used to probe this possible mechanism change. These studies will be carried out with protium and deuterium labeled reduced alternative dinucleotides and Mg(2+), Mn(2+) and Cd(2+). These studies will be followed up with secondary deuterium isotope effects using NAD-4-D and L-malate-3, 3-t2 to define the transition state structure for hydride transfer and decarboxylation (if present) steps. In addition, primary deuterium and tritium isotope effects will be used to study the reductive carboxylation reaction as well as the role of the metal ion. Studies will be extended to include the closely related isocitrate and delta-phosphoglucamate dehydrogenase to determine whether the phenomenon described above for malic enzymes is common to this class of oxidative decarboxylases. Preliminary evidence has been obtained to implicate a second metal ion (in addition to MgPPi) in the pyrophosphate phosphofructokinase (PPi-PFK) reaction. Exchange inert metal-PPi complexes will be used to test this hypothesis. The phosphoryl transfer step is rate determining for the PPi-PFK reaction and thus primary and secondary (18)O effects will be carried out using the remote label technique to probe transition state structure. The availability of a form of the ATP-PFK desensitized to hysteresis in the time courses for F6P phosphorylation and homotropic cooperativity has facilitated studies of the kinetic mechanism of regulation and the mechanism of acid-base catalysis. Initial velocity studies will be used to determine the effect of allosteric modulators along the reaction pathway, the mechanism of acid-base, catalysis, and the optimum protonation state for binding groups on reactants and effectors as well as the active and allosteric sites on enzyme. An E1cb mechanism has been proposed for aspartase in which C-N bond cleavage is rate determining. This proposed mechanism will be tested using protium washout in the NMR. In addition, the acid-base catalytic mechanism of the enzyme and optimum protonation state of binding groups will be determined.