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dc.contributor.authorManzerova, Julia
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 72-01, Section: B, page: 1320.;Advisors: Gary J. Gerfen.
dc.description.abstractRibonulceotide reductases (RNRs) are present in all cellular non-parasitic organisms. The reaction catalyzed by these enzymes is the only known source of de novo precursors for DNA replication and repair. RNRs constitute potential targets for the development of antineoplastic, antiproliferative and antimicrobial therapeutic agents.;All RNRs have in common the use of metallocofactors for the generation of a protein-based thiyl radical that is transferred to the substrate where it effects the reduction reaction. Ribonucleotide reductase from Lactobacillus leichmannii (RTPR) uses homolysis of the C-Co bond of adenosylcobalamin (AdoCbl) to produce this essential radical. AdoCbl-dependent enzymes produce greater than 11 orders of magnitude acceleration of the C-Co bond homolysis compared to the corresponding rates free in solution. In the case of RTPR, binding of an allosteric effector to the enzyme triggers C-Co bond homolysis and produces a strongly exchange-coupled thiyl radical-cob(II)alamin system. This thesis work employs multi-frequency electron paramagnetic resonance spectroscopy (EPR) in conjunction with innovative rapid freeze-quench technology (RFQ) to explore this system.;These studies have refined the size of the exchange coupling (J ex) parameter and the values of the thiyl radical g tensor. The g tensor indicates that the thiyl radical is hydrogen-bonded. Jex is much larger than previously estimated, and is larger than would be predicted for two paramagnets separated by 6.6 A in vacuo. The magnitude of the exchange coupling parameter suggests that the interaction is mediated by intervening molecular orbitals. Overall, these results represent the first experimental observation of interactions in the active site of a catalytically intact RTPR.;Multifrequency EPR approach entails examination of a sample with at least two EPR frequencies that are well separated on the spectrum (e.g. 9 GHz and 130 GHz), and offers many potential advantages over a single-frequency approach. However, 130 GHz EPR has stringent requirements for the sample form and consistency. A key part of this thesis work has been the development of a unique RFQ apparatus for the production of samples for HF EPR spectroscopy. This technological innovation is compatible with a broad range of spectroscopic techniques, and can be easily adopted by other researchers.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleRibonucleoside Triphosphate Reductase Reaction: Mechanism and Intermediates Studied with Rapid Freeze-Quench and Electron Paramagnetic Resonance Spectroscopy

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