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

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)

Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri

“…This low-potential reducing system replaced cellular-ATP-dependent reductive activation and allowed the reaction to be mediated by TMA-MT and MT2. Corrinoid-dependent methyltransferases are active in the extremely nucleophilic Co(I) state, which is converted to the methylated Co(III) state upon interaction with a methyl donor (1,2). The presence of Ti(III) citrate can result in the direct reduction of even free cobalamin to Co(I) (13).…”

Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri

“…This is consistent with the involvement of the corrinoid bound to the ␤ subunit. Corrinoid-dependent methyltransferases are in the Co(I) state following methyl group transfer (1,2,16,19,21,25,33). In this strongly nucleophilic, but oxygen-sensitive, state, they can be remethylated by the substrate.…”

Factors modulating conformational equilibria in large modular proteins: A case study with cobalamin-dependent methionine synthase

“…For instance, stopped-flow UV-visible spectrophotometric experiments involving demethylation of methylcobalamin protein with Hcy exhibit multiple phases of reaction (26), which we now interpret as arising from rate-limiting redistribution of conformations among the various forms of the protein. In addition, ligands such as AdoHcy, AdoMet, or CH 3 -H 4 folate may prove useful in obtaining x-ray crystal structures of MetH in various conformations.…”