title: Mutations in the B12-binding region of methionine synthase: how the protein controls methylcobalamin reactivity.
abstract: Vitamin B12-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine via the enzyme-bound cofactor methylcobalamin. To carry out this reaction, the enzyme must alternately stabilize six-coordinate methylcobalamin and four-coordinate cob(I)alamin oxidation states. The lower axial ligand to the cobalt in free methylcobalamin is the dimethylbenzimidazole nucleotide substituent of the corrin ring; when methylcobalamin binds to methionine synthase, the ligand is replaced by histidine 759, which in turn is linked by hydrogen bonds to aspartate 757 and thence to serine 810. We have proposed that these residues control the reactivity of the enzyme-bound cofactor both by increasing the coordination strength of the imidazole ligand and by allowing stabilization of cob(I)alamin via protonation of the His-Asp-Ser triad. In this paper we report results of mutation studies focusing on these catalytic residues. We have used visible absorbance spectroscopy and electron paramagnetic resonance spectroscopy to probe the coordination state of the cofactor and have used stopped-flow kinetic measurements to explore the reactivity of each mutant. We show that mutation of histidine 759 blocks turnover, while mutations of aspartate 757 or serine 810 decrease the reactivity of the methylcobalamin cofactor. In contrast, we show that mutations of these same residues increase the rate of AdoMet-dependent reactivation of cob(II)alamin enzyme. We propose that the reaction with AdoMet proceeds via a different transition state than the reactions with homocysteine and methyltetrahydrofolate. These results provide a glimpse at how a protein can control the reactivity of methylcobalamin.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Jarrett
authors.forename: M
authors.initials: M
authors.lastname: Amaratunga
authors.forename: C L
authors.initials: CL
authors.lastname: Drennan
authors.forename: J D
authors.initials: JD
authors.lastname: Scholten
authors.forename: R H
authors.initials: RH
authors.lastname: Sands
authors.forename: M L
authors.initials: ML
authors.lastname: Ludwig
authors.forename: R G
authors.initials: RG
authors.lastname: Matthews
mesh_terms: D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010782:Photolysis; D011485:Protein Binding; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: C019476:mecobalamin; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12