High levels of homocysteine in humans have been implicated as a factor contributing to cardiovascular disease. Methionine synthase, a methylcobalamin-dependent enzyme, converts homocysteine to methionine; as such, this enzyme is key in controlling the level of homocysteine in humans. This proposal aims to provide detailed chemical precedent and enzymatic studies focusing on one aspect of the enzyme, the coordination of histidine to cobalamin in the active site. In methionine synthase, the sixth coordinating ligand to cobalt in methylcobalamin is not the appended 5,6-dimethylbenzimidazole base, but is a histidine/imidazole residue supplied by the enzyme. A working hypothesis for the purpose of the histidine axial base is that a protonation/deprotonation cycle, in combination with varying Co-N bond lengths, is important in enhancing catalytic activity. This proposal test the hypothesis by combining chemical precedent and enzymatic studies which are designed to vary the protonation state of the axial base and the length of the Co-N bond. Thermodynamic and kinetic studies of methylcobinamide (the base-free analog of methylcobalamin) in the presence of an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be complete with and without an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be complete in the enzymatic system itself, directly probing the role of the axial base in methionine synthase.
| Dorweiler, Jeanne Sirovatka; Matthews, Rowena G; Finke, Richard G (2002) Providing a chemical basis toward understanding the histidine base-on motif of methylcobalamin-dependent methionine synthase: an improved purification of methylcobinamide, plus thermodynamic studies of methylcobinamide binding exogenous imidazole and pyri Inorg Chem 41:6217-24 |
