B12 DEPENDENT RIBONUCLEOTIDE REDUCTASE METALLOCHEMISTRY

核糖核苷酸还原酶催化核糖核苷酸还原成 脱氧核糖核苷酸;因此，它们是DNA的必需酶 所有生物体的合成和细胞生长。长期目标 这项研究的目的是获得一个分子水平的机制理解， 辅酶B 12依赖性核糖核苷酸三磷酸还原酶（RTPR）。 这种机械的理解将建立一个范式， 广泛的、仍在出现的一类基于蛋白质自由基的酶起作用， 其本质似乎是一个不寻常的，鲜为人知的激进， 链式机制这种自由基链机制也被认为是 其他13种辅酶B12依赖性和相关酶的关键， 广泛分布于人类、其他哺乳动物、植物和细菌中。的 辅酶B12辅因子，对正常成熟至关重要 红细胞，是人体内甲基丙二酰酶所需的辅因子， CoA B12不足导致恶性贫血， 包括巨幼细胞性贫血、恶性贫血和 神经系统疾病 这种生物化学上新颖的自由基链机制是由 辅酶B12的Co-C键在反应过程中的酶促均裂 具有知之甚少的蛋白质侧链"-XH"（例如，硫醇，RSH）或- X-X-（例如，二硫化物，-S-S-）位点;然后认为自由基链 是由一种我们知之甚少的酶蛋白侧链 "--X。"站点。这项建议的具体目标有四个，分为： 化学和蛋白质生物化学目标：（一）提供芬兰 辅酶B12起始步骤的化学先例， 检查辅酶B12与文献中的两个主要酶的反应， （二）对-XH或-X-X-的建议;（二）为 推定的链携带步骤，其中所得的"X. "A H. 从核糖核苷酸底物和相关的模型化合物;（iii）至 完成了一个新的，高效的，优化的开发 基于脱氧核糖核苷酸的亲和层析柱， 高度纯化的克隆和过量生产的RTPR（蛋白质纯化工作 在进行生物化学和机械研究之前， 最高可能纯度的蛋白质，可以开始），和（iv）然后开始什么 有望成为一系列迷人的蛋白质生化和 B12依赖性RTPR的机制研究。后两种RTPR蛋白 生物化学研究目标正在与 JoAnne Stubbe教授的研究小组利用他们的克隆， RTPR和关键突变体。

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.