Abstract
In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B12 cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b5. It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b5 reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b5 as a member of the reductive activation system for methionine synthase describes a function for this protein in non-erythrocyte cells. In erythrocytes, soluble cytochrome b5 functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.
Highlights
Whereas the activity of methionine synthase directly influences intracellular homocysteine concentration, its activity is itself dependent on auxiliary redox proteins that could influence homocysteine levels indirectly (Fig. 2). This is strongly supported by the existence of the cblE class of patients with an inborn error of cobalamin metabolism resulting in a functional methionine synthase deficiency, albeit the methionine synthase itself is apparently normal [13,14,15]
Mammalian methionine synthase has been the subject of intense interest since it is one of the two cellular enzymes that control homocysteine metabolism in cells
The reactivity of the cob(I)alamin intermediate results in its inadvertent escape to an oxidized and inactive enzyme form approximately once every 100 –2000 turnovers depending on the assay conditions [20, 29]
Summary
Porcine livers obtained fresh from a slaughterhouse in Crete, Nebraska, were cubed and stored frozen at Ϫ80 °C until further use. Step 1: Preparation of Homogenate—One pig liver was cubed and placed in 2 liters of 100 mM potassium Pi (pH 5.9) containing 25 mg of trypsin inhibitor, 50 mg of phenylmethylsulfonyl fluoride, 6 mg of TLCK, and 2 ml of aprotinin and was homogenized at high speed in a Waring blender for 3 ϫ 1-min bursts with 1-min intervals to prevent overheating. The column was eluted with 50 mM potassium Pi (pH 7.2) containing 0.15 M KCl. The active fractions were pooled, concentrated to ϳ1 ml, and stored at 4 °C. The anaerobic NADPH-dependent assay [15, 25] was employed, in which the reductive activation system for methionine synthase was reconstituted by adding fractions to be tested for component II activity to an assay mixture containing microsomes (0.6 mg of total protein) with component I activity and purified methionine synthase.
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