STRUCTURE/MECHANISM OF 6-PHOSPHOGLUCONATE DEHYDROGENASE

6-前列腺素脱氢酶催化β-羟基酸的氧化脱羧基 是包括苹果酸酶和异柠檬酸的一类酶的成员。 脱氢酶。从机理上讲，这类酶被认为是 催化氧化脱羧化分两步进行 在脱羧基之前，要消除的羧基的乙醇。 然而，PI实验室最近的研究表明，这一类 酶可以催化协同氧化或逐步氧化。 脱羧基依赖于所研究的酶和二核苷酸 所使用的底物。有趣的是，三维空间的比较 6-前列腺素脱氢酶、异柠檬酸脱氢酶和氨基的结构 苹果酸酶的酸序列表明，每一种都是不同的 至少在二核苷酸结合部位上与另一个不同。一个 现在有大量的机械性信息可用于 假丝酵母菌6-PGDH。然而，对于刘易斯酸是否有要求 反应的氧化脱羧基部分(酶不 需要二价金属离子)，酸碱催化和 结合基团以及每个基团对自由能的贡献 结合和/或催化作用尚不清楚。晶体结构是可用的 对于绵羊肝脏的酶，而不是对于念珠菌的酶，而是满满的 长度克隆和表达系统都不适用于这两种植物。因此， 绵羊肝酶将被克隆、测序并在制剂中表达 用于结晶和诱变，并进行了动力学研究以确定 与假丝酵母酶相比存在差异的地方(如果有)。这些 总体目标将通过以下具体目标来实现。1. 绵羊肝脏6-前列腺素脱氢酶克隆将从绵羊肝脏cDNA中分离出来 位于lambda gt10的图书馆。该资料库最初将使用以下方法进行筛选 根据已知的cDNAs序列合成寡核苷酸探针 最后通过使用大肠杆菌突变株进行代谢选择 这需要6-前列腺素脱氢酶基因才能在葡萄糖酸上生长。分离的基因片段 将对重组蛋白进行测序、表达和鉴定。2. 突变的6-PDGH酶将在没有和存在的情况下结晶 对反应物及其结构进行求解，以确定有什么不同 是由氨基酸变化引起的。这很可能是可以实现的 多数情况下采用差分法。3.绵羊的动力学研究 对肝脏进行6-前列腺素脱氢酶，以获取机制信息。 将进行选定的初始速度、pH和同位素效应研究 确定绵羊之间在质和量上的差异 肝脏6-前列腺素脱氢酶和念珠菌酶。4.定点突变 将进行以确定催化和结合基团以及 对催化的贡献。首先将重点放在酸碱上 催化基团、糖和二核苷酸结合基团。这些研究 应该会大大增加我们对6- 磷酸葡萄糖酸脱氢酶与β-羟酸氧化 一般都是脱羧酶。它们应具体规定6- 了解磷酸葡萄糖酸脱氢酶的独特机会 这种金属非依赖性氧化脱羧酶的反应动力学 就酶的结构而言。

The 6-PGDH catalyzes the oxidative decarboxylation of a beta-hydroxyacid and is a member of a class of enzymes including malic enzyme and isocitrate dehydrogenase. Mechanistically this class of enzymes is thought to catalyze the oxidative decarboxylation in two steps with oxidation of the alcohol beta to the carboxyl to be eliminated, preceding decarboxylation. Recent work from the lab of the PI, however, suggests that this class of enzymes can catalyze either a concerted or stepwise oxidative decarboxylation dependent on the enzyme studied, and the dinucleotide substrate used. Interestingly, comparison of the three dimensional structures of the 6-PGDH and the isocitrate dehydrogenase, and the amino acid sequence of the malic enzyme indicates that each of these is distinct from the other at least with respect to the dinucleotide binding site. A significant amount of mechanistic information is now available for the Candida 6-PGDH. However, whether there is a Lewis acid requirement for the oxidative decarboxylation portion of the reaction (the enzyme does not require a divalent metal ion), the identity of the acid-base catalytic and binding groups and what each contributes in terms of the free energy of binding and/or catalysis is not clear. A crystal structure is available for the sheep liver enzyme, but not for the Candida enzyme, while a full length clone and expression system is available for neither. Thus, the sheep liver enzyme will be cloned, sequenced, and expressed in preparation for crystallization and mutagenesis, and studied kinetically to determine where differences exist (if they do) compared to the Candida enzyme. These general objectives will be carried out via the following Specific Aims. 1. The sheep liver 6-PGDH clone will be isolated from a sheep liver cDNA library in lambda gt10. The library will be screened initially using oligonucleotide probes synthesized based on the known sequence of the cDNA and finally via metabolic selection using an Escherichia coli mutant strain that requires the 6-PGDH gene for growth on gluconate. The isolated cDNA will be sequenced, the recombinant protein expressed and characterized. 2. Mutant 6-PDGH enzymes will be crystallized in the absence and presence of reactants and their structures solved to determine what differences have resulted from the amino acid change. This can most likely be accomplished by the difference method in most cases. 3. Kinetic studies of the sheep liver 6-PGDH will be carried out to obtain mechanistic information. Selected initial velocity, pH, and isotope effect studies will be performed to identify the differences, qualitative and quantitative, between sheep liver 6-PGDH and the enzyme from Candida. 4. Site-directed mutagenesis will be carried out to identify catalytic and binding groups and the contribution to catalysis. Emphasis will initially be placed on acid-base catalytic groups, sugar and dinucleotide binding groups. These studies should significantly increase our understanding of the mechanism of 6- phosphogluconate dehydrogenase, and beta-hydroxyacid oxidative decarboxylases in general. They should specifically provide for the 6- phosphogluconate dehydrogenase the unique opportunity to understand the kinetics of the reaction of this metal-independent oxidative decarboxylase in terms of the enzyme's structure.