Purification And Properties Of Methionine Synthase From Escherichia Coli B (cobalamine, Nitrous Oxide).

Abstract

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, E. C. 2.1.1.13) was purified to homogeneity from Escherichia coli B. Subunit and apparent molecular weights of purified enzyme were respectively 133,300 and 179,000, indicating the enzyme consisted of one polypeptide chain. The oxidation state of cobalamin in isolated enzyme was found to be cob(II)alamin, determined by EPR and visible absorbance spectra. Copper and iron were also associated with purified enzyme. Methionine synthase was irreversibly inhibited during in vitro turnover in nitrous oxide equilibrated buffer; enzyme-bound cob(I)alamin, transiently formed during turnover, reacted with nitrous oxide to generate inactive cob(II)alamin enzyme. There was spectrophotometric evidence for the partial loss of enzyme-bound cobalamin during inactivation, possibly caused by the homolytic cleavage of nitrous oxide to N(,2) and hydroxyl radical and the subsequent addition of the radical to the corrin ring. Reaction of hydroxyl radical with active center residues might also account for irreversible activity loss. The prosthetic group of inactive cob(II)alamin enzyme was methylated by S-adenosylmethionine in the presence of NADPH and two small flavoproteins from E. coli, supporting the proposed priming mechanism of inactive enzyme to the active methylcobalamin form. The EPR spectrum of cob(II)alamin enzyme was a combined spectrum of base-off cob(II)alamin and superoxo-cob(III)alamin; methylation of the cob(II)alamin resulted in loss of superoxo-cob(III)alamin signal and an overall loss of spin. The stereochemistry of the methyl transfer catalyzed by methionine synthase was determined. Transfer of the methyl group from chiral 5-methyltetrahydrofolate to homocysteine resulted in net retention of configuration of the methyl group, accompanied by about 50% racemization.