ENZYME CATALYSIS OF ELECTRON AND GROUP TRANSFER

丙酮酸脱氢酶复合物的四级结构 2-酮戊二酸酯复合物（KDH复合物）。大肠杆菌将是 电镜观察分析。 结构模型最近 使用扫描透射电子显微镜 显微镜（STEM）和径向质量分析将进一步测试。 类似的方法也将应用于地层的四级结构分析 KDH复合物中排列着2-酮戊二酸脱氢酶（E1） 立方核心蛋白二氢聚反式琥珀酰（E2）的亚基 应该不同于PDH中相应蛋白质所观察到的 复杂. 与二氢硫辛酰基连接的硫辛酰基的位置 相对于丙酮酸的PDH复合物的转乙酰（E2）组分 脱氢酶（E1）和二氢硫辛酰脱氢酶（E3）亚基将是 通过用11个金簇标记二氢硫辛酰基部分建立 复合体和使用STEM进行径向质量分析。 十一金 通过单官能酰化和烷基化制备的簇合物 试剂将用于标记PDH复合物中的硫辛酰胺部分。 的 这些标签相对于E1和E3的位置将通过径向映射 使用STEM进行质量分析。 PDH复合物中E1和E2之间的还原酰化反应机理 将被调查，关于机械的问题， 电子转移和基团转移的关系。 为此目的， 将制备2-乙酰基-TPP，并研究其酶和非酶性质 研究了 用[2- 14 C]丙酮酸盐和PDH复合物的捕获实验 将在稳态下进行[1- 14 C]乙酰基-TTP的搜索。 一 2-（1-羟基亚乙基）-TTY的自由基阳离子形式将在 E1、铁氰化物和丙酮酸盐的实验。 自旋捕获实验 也将进行。 PDH复合物的自杀失活 3-氟丙酮酸和丙酮酸加O2将被表征以确定 失活机制。 赖氨酸2，3-氨基变位酶将从梭菌中纯化，并进一步 其特征在于，特别涉及其金属离子含量和 可能存在Fe-S簇。 反应机理将是 通过确定氚动力学同位素效应的研究， β-[2- 3 H]赖氨酸的反应。 磷酸吡哆醛参与 也将研究这种B12样反应。

The quaternary structures of pyruvate dehydrogenase complex (PDH complex) and 2-ketoglutarate dehydrogenas complex (KDH complex) from E. coli will be further anlayzed by electron microscopy. The structural model recently developed for the PDH complex using scanning transmission electron microscopy (STEM) and radial mass analysis will be further tested. Similar methods will be applied to quaternary structural analysis of the KDH complex in which the arrangement of 2-ketoglutarate dehydrogenase (E1) subunits about the cubic core protein dihydolipoly transsuccinylas (E2) should differ from that observed for the corresponding proteins in the PDH complex. The locations of the lipoyl groups attached to the dihydrolipoyl transacetylas (E2) component of PDH complex relative to pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) subunits will be established by labeling the dihydrolipoyl moieties with undecagold cluster complexes and carrying out radial mass analysis using STEM. Undecagold cluster complexes prepared as monofunctional acylating and alkylating agents will be used to label lipoamide moieties in the PDH complex. The positions of these labels relative to E1 and E3 will be mapped by radial mass analysis using STEM. The mechanism of reductive acylation between E1 and E2 of the PDH complex will be investigated, with reference to the question of the mechanistic relationship between electron transfer and group transfer. To this end, 2-acetyl-TPP will be prepared and its enzymatic and nonenzymatic properties investigated. Trapping experiments with [2-14C]pyruvate and PDH complex will be carried out in search of [1-14C]acetyl-TTP in the steady state. A radical cation form of 2(1-hydroxyethylidene)-TTY will be sought in experiments with E1, ferricyanide and pyruvate. Spin trapping experiments will also be carried out. Suicide inactivation of the PDH complex by 3-fluoropyruvate and by pyruvate plus 02 will be characterized to determine the inactivation mechanisms. Lysin 2,3-aminomutase will be purified from Clostridia and further characterized, with special reference to its metal ion content and the possible presence of a Fe-S cluster. The reaction mechanism will be investigated by determining the tritium-kinetic isotope effect for the reaction of beta-[2-3H]lysine. The involvement of pridoxal phospate in this B12-like reaction will also be investigated.