Allosteric inhibition of methylenetetrahydrofolate reductase by adenosylmethionine. Effects of adenosylmethionine and NADPH on the equilibrium between active and inactive forms of the enzyme and on the kinetics of approach to equilibrium.
In this paper we report on the allosteric regulation of the dimeric flavoprotein methylenetetrahydrofolate reductase (E.C. 1.5.1.20) by the inhibitor, AdoMet, and by one of the substrates, NADPH. These metabolites play antagonistic roles in this regulation, with NADPH recruiting active forms of the enzyme and AdoMet recruiting inactive forms. At high NADPH concentrations, activity dependence on AdoMet is sigmoidal, indicating cooperativity. The kinetics of inhibition induced by AdoMet are slow enough to be studied by conventional methods and exhibit marked biphasicity. Both the extents and rates of these phases are again affected antagonistically by the ligands, AdoMet increasing the extent of the faster phase, and NADPH decreasing the extent of the faster phase and the rate of the slower phase. We present a model consistent with these observations. Our model postulates two states of the enzyme, R and T. NADPH and AdoMet exhibit antagonistic binding to a given subunit, so that occupancy by one ligand decreases or abolishes affinity for the other ligand. However, within a given state, the subunits do not interact with each other, so the ligation of one does not affect the affinities of its neighbor. R-T transitions occur between all similarly ligated states. The ligands have different affinities for the R and T states, and AdoMet binding to a given subunit is measurably slow. This model predicts the observed features of the equilibrium and kinetic data noted above. We also present a system for simulation of reaction schemes in which each step is pseudo first order that is fast and versatile enough to allow least squares fitting of microscopic rate constants to kinetic data.
D007700:Kinetics; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008956:Models, Chemical; D009249:NADP / Q000494:pharmacology*; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors*; D012436:S-Adenosylmethionine / Q000494:pharmacology*
D009249:NADP; D012436:S-Adenosylmethionine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)