STRUCTURE AND MECHANISM OF NAD-MALIC ENZYME

这项研究的主要目的是阐明 蛔虫NAD-苹果酸酶的作用机制 suum。NAD-苹果酸酶是一类吡啶-核苷酸- 依赖酶催化β- 羟基酸基于该类中t种不同酶之间的同源性 吡啶连接的氧化脱羧酶， 磷酸葡萄糖酸脱氢酶（6-PGDH）属于结构类型 与金属依赖性异柠檬酸盐（ICDH）不同， 异丙苹果酸（IMDH）脱氢酶，两者均不同于苹果酸脱氢酶 酶，其还需要二价金属离子活化剂。三 已经解决了6-PGDH和ICDH的三维结构，但没有 苹果酸酶的结构是可利用的。目前我们知道的很多 男性酶的机制，包括信息的性质， 氧化和脱羧的过渡态。仍然 有趣的问题，然而，关于氧化和 脱羧，以及酶结构与 苹果酸酶亚类的催化作用。在本申请中， 建议继续研究苹果酸酶的作用机制。的 总目标将通过以下具体目标实现。 通过动力学同位素效应的机制研究。 一些问题 关于过渡态结构和耦合的性质， 氧化和脱羧。多重初级氘/β-次级氘 氚同位素效应与标记在C-3的苹果酸将被用来 区分分步和协同氧化脱羧， 二核苷酸底物被改变。α-次级氚同位素 NAD烟酰胺环4位氚的影响 将进行测量，以获得有关转让程度的信息， 氢化物处于氧化的过渡态。以确定是否 氢隧穿和耦合运动发生时，D/T方法 Saunders/Klinman将用于测定初级和次级氚 方面的影响.通过定点突变的结构-活性。 定点 诱变将用于探测金属、苹果酸盐和NAD结合 位置，一般酸的身份一般碱催化基团， 氢化物转移的反应坐标上的残基 反应，以及建立活性、催化 酶的构象。NAD-苹果酸酶的结构。三 获得蛔虫NAD-苹果酸酶的三维结构 与亚拉巴马大学的拉里·德卢卡斯博士合作， 伯明翰此外，光谱探针如荧光和CD将 与NAD一起使用，3-APAD在不存在和存在Mg 2的情况下结合， 苹果酸。此外，试验已经在进行中，以结晶E：3- APAD复合体。很少有人能观察到真正的过渡态变化 在酶促反应中。在苹果酸酶的情况下， 已经或可能已经在两种化学物质的偶联中被诱导， 步骤，并有证据表明，增加氢隧道与 金属离子活化剂的变化。

The primary objective of this research proposal is to elucidate the mechanism of the NAD-Malic enzyme from the parasitic nematode, Ascaris suum. NAD-Malic enzyme is a member of a class of pyridine-nucleotide- dependent enzymes that catalyze the oxidative decarboxylation of beta- hydroxy acids. Based on homology amongst t different enzymes in the class of pyridine-linked oxidative decarboxylases, the metal-independent phosphogluconate dehydrogenase (6-PGDH) is in a structural class different from that of the metal-dependent isocitrate (ICDH) and isopropylmalate (IMPDH) dehydrogenases, and both differ from the malic enzyme, which also requires a divalent metal ion activator. Three dimensional structures have been solved for 6-PGDH an ICDH, but no structure is yet available for a malic enzyme. Much is presently known of the mechanism of male enzyme, including information on the nature of the transition states for oxidation and decarboxylation. There still interesting questions, however, regarding the coupling of oxidation and decarboxylation, and the relationship between enzyme structure and catalysis for the malic enzyme subclass. In the present application, it is propose to continue studies on the mechanism of the malic enzyme. The overall goal will be achieved via the following Specific Aims. Mechanistic Studies via Kinetic Isotope Effects. A number of questions remain concerning transition state structure and the nature of coupling oxidation and decarboxylation. Multiple primary deuterium/beta-secondary tritium isotope effects with label at C-3 of malate will be used to distinguish between stepwise and concerted oxidative decarboxylation as the dinucleotide substrate is changed. alpha-Secondary tritium isotope effects with tritium at the 4 position of the nicotinamide ring of NAD will be measured to obtain information on the extent of transfer of the hydride in the transition state for oxidation. To determine whether hydrogen tunneling and coupled motion occurs, the D/T method of Saunders/Klinman will be used to determine primary and secondary tritium effects. Structure-Activity via Site-Directed Mutagenesis. Site-directed mutagenesis will be used to probe the metal, malate and NAD binding sites, the identity of the general acid an general base catalytic groups, residues lining the reaction coordinate for the hydride transfer reaction, and an residues required for setting up the active, catalytic conformation of the enzyme. Structure of the NAD-Malic Enzyme. The three dimensional structure of the Ascaris NAD-Malic enzyme will be obtained in collaboration with Dr. Larry DeLucas at the University of Alabama at Birmingham. In addition, spectral probe such as fluorescence, and CD will be used with NAD, and 3-APAD bound in the absence and presence of Mg2 and malate. In addition, trials are already underway to crystallize the E:3- APAD complex. Very seldom doe one observe true transition state changes in enzymatic reactions. In the case of the malic enzyme, changes either have or may have been induced in the coupling of two of the chemical steps, and there is evidence of an increase in hydrogen tunneling with change in metal ion activator.