Participation of Cob(I)alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis

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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.degree. 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). Enzyme-bound cob(I)alamin was also shown to form rapidly (140 s-1) when homocysteine was mixed with methylated enzyme, thus establishing its kinetic relevance as an intermediate. A minimal kinetic mechanism that accommodates the steady-state data and the measured rate constants was employed to simulate the kinetic behavior of the system. The simulations made very different predictions depending on whether the prosthetic group was in the cob(I)alamin or methylcobalamin state in "free" enzyme and yielded an excellent fit to the real data only when enzyme containing bound methylcobalamin was employed as the starting enzyme. From these studies it was concluded that methylcobalamin-containing methionine synthase is the "free" form of the enzyme, to which the substrates bind in an ordered sequential fashion, and that enzyme-bound cob(I)alamin is a kinetically competent intermediate.