B12 DEPENDENT RIBONUCLEOTIDE REDUCTASE METALLOCHEMISTRY

B12 依赖性核糖核苷酸还原酶金属化学

• 批准号: 2137834
• 负责人: RICHARD G FINKE
• 金额: $ 16.32万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137834
• 关键词: affinity chromatography; catalyst; cobalt; deoxyribonucleotides; disulfide bond; electrospray ionization mass spectrometry; enzyme mechanism; enzyme substrate; erythrocytes; glutathione; homocysteine; hydrogen bond; hydroxymethyltransferases; iron; methylmalonyl coA epimerase; molecular biology; molecular site; protein purification; proteins; reduction; vitamin B12 coenzyme; zinc

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides; they are, therefore, essential enzymes for DNA synthesis and cell growth in all living organisms. The long term objective of this research is to obtain a molecular-level mechanistic understanding of Coenzyme B 12-dependent Ribonucleotide Triphosphate Reductase (RTPR). Such mechanistic understanding will establish a paradigm by which a very broad, and still emerging, class of protein-radical-based enzymes operate, the essence of which appears to be an unusual, poorly understood radical- chain mechanism. This radical-chain mechanism is also thought to be the key to the other thirteen coenzyme B12-dependent and related enzymes that are widely distributed in humans, other mammals, plants and bacteria. The coenzyme B12 cofactor, essential for the normal maturation of erythrocytes, is a required cofactor in man for the enzyme methylmalonyl- CoA mutase. Insufficient B12 results in pernicious anemia with clinical features that include megaloblastic anemia, malignant anemia, and neurological disorders. This biochemically novel radical-chain mechanism is initiated by the enzyme-accelerated homolysis of Coenzyme B12's Co-C bond during a reaction with a poorly understood protein side-chain "-XH" (e.g., thiol, RSH) or - X-X- (e.g., disulfide, -S-S-) site; the radical chain is then thought to be propagated by an even more poorly understood enzyme protein side-chain "-X." site. The specific aims of this proposal are fourfold, and divide up into chemical and protein biochemical objectives: (i) to provide finn chemical precedent for (or against) the Coenzyme B12 initiation step by examining the reaction of Coenzyme B12 with the literature's two main suggestions for -XH or -X-X-; (ii) to provide firm precedent for the putative chain-carrying step in which the resultant "X." abstracts a H. from ribonucleotide substrates and related model compounds; (iii) to finish the development of a new, high-efficiency, and optimized deoxyribonucleotide-based affinity chromatography column for obtaining highly purified cloned and overproduced RTPR (protein purification work necessary before biochemical and mechanistic studies, which require the highest possible purity protein, can begin), and (iv) to then begin what promises to be a long series of fascinating protein biochemical and mechanistic studies of B12-dependent RTPR. These latter two RTPR protein biochemical research objectives are being pursued in collaboration with Professor JoAnne Stubbe's research group using their cloned, overproduced RTPR and key mutants.

核糖核苷酸还原酶催化核糖核苷酸还原为 脱氧核糖核苷酸；因此，它们是DNA的必需酶 在所有生物中的合成和细胞生长。长期目标 这项研究的目的是获得分子水平的机理理解 辅酶B12依赖的核糖核酸三磷酸还原酶(RTPR)。 这种机械性的理解将建立一种范式，通过这种范式， 一类广泛的、仍在不断涌现的以蛋白质为基础的酶 它的本质似乎是一种不同寻常的，鲜为人知的激进分子- 链条机构。这种自由基链机制也被认为是 其他13种依赖辅酶B12和相关酶的关键 广泛分布于人类、其他哺乳动物、植物和细菌中。这个 辅酶B12辅因子，正常成熟所必需的 红细胞，是人体甲基丙二酰基酶所必需的辅因子- 辅酶A变位酶。B12不足导致恶性贫血 特征包括巨幼细胞性贫血、恶性贫血和 神经紊乱。 这种生化上新颖的自由基链机制是由 酶促反应过程中辅酶B12‘S Co-C键的均裂 具有一个鲜为人知的蛋白质侧链“-XH”(例如，硫醇、RSH)或- X-X-(例如，二硫化物，-S-S-)位；然后认为自由基链 是由一种更鲜为人知的酶蛋白侧链繁殖的 “-X。”地点。这项建议的具体目标有四个，并分为四个部分 化学和蛋白质生化目标：(I)为芬兰人提供 辅酶B12引发(或反对)的化学先例 考察辅酶B12与文献中两个主要成分的反应 建议-XH或-X-X-；(Ii)为 假定的链携带步骤，在该步骤中，所得到的“X。”提炼出一个H。 来自核糖核苷酸底物和相关模型化合物； 完成新的、高效的、优化的开发 一种基于脱氧核糖核苷酸的亲和层析柱 高纯度克隆和高产量RTPR(蛋白质纯化工作 在生化和机械学研究之前是必要的，这需要 最高纯度的蛋白质，可以开始)，和(Iv)然后开始什么 有望成为一长串令人着迷的蛋白质生化和 B12依赖的RTPR的机制研究。后两种RTPR蛋白 正在与以下机构合作实现生化研究目标 JoAnne Stubbe教授的研究小组使用他们的克隆，过度生产 RTPR和关键突变体。