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.
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