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PMID:2271698

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Citation

Banerjee, RV, Frasca, V, Ballou, DP and Matthews, RG (1990) Participation of cob(I) alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis. Biochemistry 29:11101-9

Abstract

The kinetic mechanism of the reaction catalyzed by cobalamin-dependent methionine synthase from Escherichia coli K12 has been investigated by both steady-state and pre-steady-state kinetic analyses. The reaction catalyzed by methionine synthase involves the transfer of a methyl group from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The postulated reaction mechanism invokes an initial transfer of the methyl group to the enzyme to generate enzyme-bound methylcobalamin and tetrahydrofolate. Enzyme-bound methylcobalamin then donates its methyl group to homocysteine to generate methionine and cob(I)alamin. The key questions that were addressed in this study were the following: (1) Does the reaction involve a sequential or ping-pong mechanism? (2) Is enzyme-bound cob(I)alamin a kinetically competent intermediate? (3) If the reaction does involve a sequential mechanism, what is the nature of the "free" enzyme to which the substrates bind; i.e., is the prosthetic group in the cob(I)alamin or methylcobalamin state? Both the steady-state and rapid reaction studies were conducted at 25 degrees C under anaerobic conditions. Initial velocity analysis under steady-state conditions revealed a family of parallel lines suggesting either a ping-pong mechanism or an ordered sequential mechanism. Steady-state product inhibition studies provided evidence for an ordered sequential mechanism in which the first substrate to bind is methyltetrahydrofolate and the last product to be released is tetrahydrofolate. Pre-steady-state kinetic studies were then conducted to determine the rate constants for the various reactions. Enzyme-bound cob(I)alamin was shown to react very rapidly with methyltetrahydrofolate (with an observed rate constant of 250 s-1 versus a turnover number under maximal velocity conditions of 19 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Links

PubMed

Keywords

5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/metabolism; Escherichia coli/enzymology; Kinetics; Models, Theoretical; Protein Binding; Time Factors; Vitamin B 12/analogs & derivatives; Vitamin B 12/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:METH

enables

GO:0031419: cobalamin binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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