KINETIC AND CHEMICAL MECHANISMS OF ENZYMES

这项建议的总体目标是了解酵素 机制。努力集中在三个酶系统上，包括 苹果酸酶、磷酸果糖激酶和天冬氨酸酶。三步化学反应 苹果酸酶的作用机制已被提出，其中苹果酸是第一位的 氧化成草酸乙酸酯，草酸乙酸酯中间体被脱羧基 烯醇式丙酮酸，后者然后互变异构化为丙酮酸。近期 用替代二核苷酸底物获得的证据表明 有两种可能性。首先，有一个机制的变化， 苹果酸氧化脱羧制丙酮酸烯醇酯 协调机制。其次，存在二次(13)C同位素效应 在苹果酸氧化成草酸乙酸酯中间体的过程中。这个 草酸乙酯中间体在E：NADH：Mg：草酸乙酯中的分配 复合体，向苹果酸和丙酮酸将被用来探索这一可能性 机制的改变。这些研究将与Protium和 氚标记的还原二核苷酸和镁(2+)、锰(2+)和 Cd(2+)。这些研究将用二次氚同位素进行后续研究。 用NAD-4-D和L-苹果酸-3，3-T2定义过渡态的效果 氢化物转移和脱羧化(如果存在)步骤的结构。在……里面 此外，初级氚和氚的同位素效应将被用于 研究了还原羧化反应以及金属的作用 离子。研究将扩大到包括密切相关的异柠檬酸和 用Delta-磷酸葡萄糖脱氢酶来判断现象是否存在 上面描述的苹果酸酶对于这类氧化是共同的 脱羧酶。已获得初步证据，表明 焦磷酸盐中的第二金属离子(除MgPPI外) 磷酸果糖激酶(PPI-PFK)反应。交换惰性金属-PPI络合物 将被用来检验这一假设。磷酰化转移步骤是速率 PPI-PFK反应及(18)O的测定 效果将使用远程标签技术进行探测 过渡态结构。一种形式的ATP-PFK的可用性 F6P磷酸化和F6P的时程对滞后不敏感 各向同性的协作性促进了动力学机制的研究 的调节和酸碱催化的机制。初速 研究将用于确定变构调节剂的效果 介绍了反应途径、酸碱机理、催化作用和反应机理。 反应物和效应物As上结合基团的最佳质子化状态 以及酶上的活性部位和变构部位。E1cb机制具有 提出了C-N键裂解为速率决定的天冬氨酸氨基转移酶。 这一提出的机制将在核磁共振中使用Proashout进行测试。 此外，还探讨了酶的酸碱催化机理和最适条件 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.