Joseph T. Jarrett

Purification and assay of cobalamin-dependent methionine synthase from Escherichia coli

Publisher Summary This chapter presents the purification and assay of cobalamin-dependent methionine synthase from Escherichia coli . The assay, using radioactively labeled methyltetrahydrofolate, modified from that described by Taylor and Weissbach, is most suitable for measuring activity in crude extracts, while the nonradioactive assay is less expensive and more convenient for use with purified enzyme. Cobalamin-dependent methionine synthase, or 5-methyltetrahydrofolate-homocysteine S-methyltransferase (EC 2.1.1.13), from Escherichia coli is a 136,902-Da peptide containing one molecule of bound cobalamin. The enzyme catalyzes the transfer of a methyl group from methyltetrahydrofolate to the cob(I)alamin form of the cofactor to form methylcobalamin and tetrahydrofolate and then transfers the methyl group from methylcobalamin to homocysteine, forming methionine and regenerating the cob(I)alamin form of the enzyme. Cob(I)alamin is not only a powerful nucleophile but also a potent reductant, capable of reducing the protons of solvent to hydrogen gas. This form of the enzyme occasionally becomes oxidized to an inactive cob(II)alamin species. Return of this species to the catalytic cycle requires a reductive methylation, in which the methyl group is supplied by adenosylmethionine rather than methyltetrahydrofolate. In Escherichia coli , the electrons for reductive activation are supplied by reduced flavodoxin. For in vitro assays, other reducing systems are often used; most frequently electrons for reductive activation are supplied by using dithiothreitol or 2-mercaptoethanol and catalytic amounts of hydroxocobalamin.

A protein radical cage slows photolysis of methylcobalamin in methionine synthase from Escherichia coli

Methionine synthase from Escherichia coli is a B12-dependent enzyme that utilizes a methylcobalamin prosthetic group. In the catalytic cycle, the methyl group of methylcobalamin is transferred to homocysteine, generating methionine and cob(I)-alamin, and cob(I)alamin is then remethylated by a methyl group from methyltetrahydrofolate. Methionine synthase occasionally undergoes side reactions that produce the inactive cob(II)alamin form of the enzyme. One such reaction is photolytic homolysis of the methylcobalamin C-Co bond. Binding to the methionine synthase apoenzyme protects the methylcobalamin cofactor against photolysis, decreasing the rate of this reaction by approximately 50-fold. The X-ray structure of the cobalamin-binding region of methionine synthase suggests how the protein might protect the methylcobalamin cofactor in the resting enzyme. In particular, the upper face (methyl or beta face) of the cobalamin cofactor is in contact with several hydrophobic residues provided by an alpha-helical domain, and these residues could slow photolysis by caging the methyl radical and favoring recombination of the CH3./cob(II)alamin radical pair. We have introduced mutations at three positions in the cap domain; phenylalanine 708, phenylalanine 714, and leucine 715 have each been replaced by alanine. Calculations based on the wild-type structure predict that two of these three mutations (Phe708Ala and Leu715Ala) will increase solvent accessibility to the methylcobalamin cofactor, and in fact these mutations result in dramatic increases in the rate of photolysis. The third mutation, Phe714Ala, is not predicted to increase the accessibility of the cofactor and has only a modest effect on the photolysis rate of the enzyme. These results confirm that the alpha-helical domain covers the cofactor in the resting methylcobalamin enzyme and that residues from this domain can protect the enzyme against photolysis. Further, we show that binding the substrate methyltetrahydrofolate to the wild-type enzyme results in a saturable increase in the rate of photolysis, suggesting that substrate binding induces a conformational change in the protein that increases the accessibility of the methylcobalamin cofactor.