STRUCTURE/MECHANISM OF 6-PHOSPHOGLUCONATE DEHYDROGENASE

6-PGDH催化β-羟基酸的氧化脱羧 并且是包括苹果酸酶和异柠檬酸盐在内的一类酶的成员 脱氢酶。 从机理上讲，这类酶被认为是 分两步催化氧化脱羧， 在脱羧之前，羧基的β位醇被除去。 然而，PI实验室最近的工作表明，这类 酶可以催化协同或逐步氧化 脱羧依赖于酶的研究，和二核苷酸 使用的衬底。 有趣的是， 6-PGDH和异柠檬酸脱氢酶的结构，以及氨基 苹果酸酶的酸序列表明，这些酶中的每一种都是不同的 至少相对于二核苷酸结合位点是彼此不同的。 一 大量的机械信息现在可用于 念珠菌6-PGDH。 然而，是否有一个刘易斯酸的要求， 反应的氧化脱羧部分（酶不 需要二价金属离子）、酸-碱催化剂的特性以及 结合基团以及每个结合基团在自由能方面的贡献 结合和/或催化作用尚不清楚。 晶体结构可用 羊肝酶，但不是念珠菌酶，而一个完整的 长度克隆和表达系统对两者都是不可用的。 因此 将羊肝酶进行克隆、测序，并在制备中表达 结晶和诱变，并进行动力学研究，以确定 其中与念珠菌酶相比存在差异（如果存在的话）。 这些 将通过以下具体目标实现总体目标。 1. 将从绵羊肝脏cDNA中分离绵羊肝脏6-PGDH克隆 lambda gt 10中的库。 文库将首先使用 基于cDNA的已知序列合成的寡核苷酸探针 最后通过使用大肠杆菌突变株的代谢选择 需要6-PGDH基因才能在葡萄糖酸盐上生长 分离的cDNA 将被测序，重组蛋白表达和表征。 2. 突变体6-PDGH酶将在不存在和存在下结晶， 反应物和它们的结构解决，以确定有什么不同， 这是由氨基酸的变化引起的。 这很有可能通过 在大多数情况下，采用差分法。 3. 绵羊的动力学研究 将进行肝脏6-PGDH以获得机制信息。 将进行选定的初始速度、pH值和同位素效应研究 以确定绵羊之间的差异，定性和定量， 肝脏6-PGDH和念珠菌的酶。 4. 定点诱变 将进行，以确定催化和结合基团和 催化的贡献。 重点将首先放在酸碱 催化基团、糖和二核苷酸结合基团。 这些研究 应该大大增加我们对6- 磷酸葡萄糖酸脱氢酶和β-羟基酸氧化 脱羧酶。 他们应该具体规定6- 磷酸葡萄糖酸脱氢酶的独特机会，了解 这种非金属依赖性氧化脱羧酶的反应动力学 在酶的结构方面。

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.