A program of study is proposed to determine the mechanism by which an enzyme requiring 5'-deoxy-adenosylcobalamin (AdoCbl, coenzyme B12) accelerates the rate of homolysis of the carbon-cobalt bond of AdoCbl by 9 to 12 orders of magnitude. The enzyme to be studied is the ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Recent work from the principal investigator's laboratory has shown that catalysis of AdoCbl Co-C bond homolysis by RTPR occurs with a large (14 kcal mol-1) decrease in the enthalpy of activation but no change in the entropy of activation, relative to non-enzymatic, thermal Co-C bond homolysis. Three hypotheses for the enthalpic catalysis of AdoCbl homolysis will be investigated: (1) Stabilization of the transition state for Co-C bond homolysis by partial transfer of a hydrogen atom from the essential active site cysteine, Cys408; (2) Ground state Co-C bond distortion by interaction of the active site of RTPR with the Ado ligand of AdoCbl; (3) Mechanochemical triggering, in which enzymatic compression of the axial Co-N bond of AdoCbl leads either to ground state destabilization of the Co-C bond, or to electronic stabilization of the transition state by increased Co-Nax orbital overlap. The following experiments are designed to investigate these possibilities: (1) Studies of structure-activity relationships of AdoCbl structural analogs which are partially active coenzymes for RTPR, including X-ray crystallography, NMR-restrained molecular modeling, and kinetic studies of the RTPR-induced Co-C bond homolysis; (2) Studies of the effect of complexation to RTPR on the 13C NMR chemical shift and 1H- 13C coupling constants of AdoCbl enriched in 13C in the cobalt-bound carbon, and on the 15N NMR chemical shift of AdoCbl's individually enriched in 15N in each of the Ado nitrogens and in the coordinating nitrogen of the axial nucleotide; (3) Studies of AdoCbl's with unnatural lower axial nucleotides of lower steric bulk (such as benzimidazole and imidazole) than the natural axial nucleotide, including X-ray crystallography, NMR-restrained molecular modeling, and complete kinetic studies (including temperature dependence) of the RTPR-induced and non-enzymatic thermal homolysis of the Co-C bond; and (4) Studies of models of the active site of RTPR containing an intramolecular thiol function attached to AdoCbl by tethers of varying lengths. In addition, the possibility that the active species of RTPR is actually a dimer will be investigated by determining the effect of protein concentration on the observed binding constants for AdoCbl and the allosteric effector dGTP.
| Chandra, Tilak; Brown, Kenneth L (2008) Vitamin B(12) and alpha-Ribonucleosides. Tetrahedron 64:9-38 |
| Brown, Kenneth L; Li, Jing; Zou, Xiang (2006) NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii. Inorg Chem 45:9172-4 |
| Brown, Kenneth L; Chandra, Tilak; Zou, Shawn et al. (2005) Regioselective glycosylation: synthesis of alpha-indoline nucleosides. Nucleosides Nucleotides Nucleic Acids 24:1147-65 |
| Brown, K L; Zou, X (1999) Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus J Inorg Biochem 77:185-95 |
