Cobalamin is a derivative of vitamin B12 and has been recruited as a cofactor by two known mammalian enzymes: methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). MCM catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, and represents an intermediate step in the catabolism of branched chain amino acids and odd chain fatty acids. Impaired mutase activity leads to methylmalonic acidemia, and, in severe cases to neonatal death. Methionine synthase catalyzes a transmethylation reaction between methyltetrahydrofolate and homocysteine to generate methionine, which serves both in protein synthesis and is converted to S-adenosylmethionine, a vital cellular methyl group donor. In mammals, methionine synthase plays a seminal role in both folate and sulfur metabolism. Dysfunction of methionine synthase results in megaloblastic anemia. This proposal focuses on the mechanistic enzymology of mammalian cobalamin-dependent enzymes. Chemically, cobalamin is a novel cofactor, and catalyzes distinct biochemical transformations in association with the two proteins. Thus, in association with MCM cobalamin effects an intramolecular 1,2 rearrangement reaction whereas on methionine synthase the cofactor mediates an intermolecular transfer of a methyl group. We propose to conduct the following specific experiments with the mutase: (1) EPR spectroscopy and rapid reaction kinetic analysis will be employed to determine if Co-C bond homolysis initiates the isomerization reaction, (2) rapid reaction kinetic analysis will be employed to establish the nature of the migrating group (i.e. whether it is a carbanion, a carbocation, a radical or an organocobalt adduct), (3) heavy atom and deuterium kinetic isotope effect studies will be conducted to determine the extent to which hydrogen transfer is rate limiting, and (4) the equilibrium midpoint potentials of the cob(II)alamin/cob(I)alamin and cob(II)alamin/cob(III)alamin redox couples will be measured by EPR spectroelectrochemical titrations. We propose to conduct the following studies with MS: (1) purify MS from pig liver to homogeneity, and generate both N-terminal amino acid sequence information and anti-MS antibodies, (2) clone the gene encoding MS from a human liver cDNA library, determine the nucleotide sequence and express the gene product in E. coli or yeast, (3) determine the structure and function of iron by metal replacement and spectroscopic studies involving EPR, EXAFS and Mossbauer spectroscopy, and (4) determine the components of the activation system. Characterization of the native enzymes is seen as a prelude to the biochemical characterization of the impairments in cell lines from patients born with inborn errors of metabolism affecting one of these enzymes.
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