| Vascular disease is multifactorial and not all risk factors have been identified. A substantial number of patients have elevated levels of plasma total homocysteine (Hcy), which is now considered a risk factor for cardiovascular disease, stroke, and Alzheimer's disease. Endothelial damage may be caused by Hcy-induced vascular oxidative stress. Hcy is not a dietary amino acid, but is an important branch-point intermediate of methionine metabolism. Betaine-homocysteine S-methyltransferase (BHMT) is one of two enzymes capable of remethylating Hcy to methionine. We report that BHMT is susceptible to conformation-dependent oxidative inactivation. Two oxidants, methyl methanethiosulfonate (MMTS) and hydrogen peroxide (H2O2), cause a loss of BHMT's catalytic Zn and a correlative loss of activity. Addition of beta-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. Circular dichroism and glutaraldehyde crosslinking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by carboxybutylhomocysteine (CBHcy), a bisubstrate analog, but methionine alone has no protective effect. Partial digestion of ligand-free BHMT with trypsin produces two large peptides, excising a seven residue peptide within loop L2. CBHcy but not methionine binding slows proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine binding site, and that this conformational change and/or occupancy of both ligand binding sites protects the enzyme from oxidative inactivation. In addition to the direct effect of oxidative stress, we studied a possible indirect effect by phosphorylation by protein kinase C (PKC). PKC has been implicated in one mechanism by which Hcy causes endothelial injury. We demonstrate BHMT phosphorylation by PKC in vitro, which is attenuated by the presence of CBHcy. BHMT activity is increased in liver extracts treated with alkaline phosphatase. These data suggest direct and indirect pathways to downregulate BHMT and consequently shunt Hcy toward synthesis of the antioxidant glutathione to reduce oxidative stress. Information about how the cell combats oxidative stress by regulating intracellular thiols may lead to preventative measures and potential targets of treatment options for vascular diseases. |
