Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase.
Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH(3)-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE.CH(3)-H(4)folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH(3)-H(4)folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions.
Taurog
Rebecca E
RE
Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Jakubowski
Hieronim
H
Matthews
Rowena G
RG
D002151:Calorimetry; D002384:Catalysis; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D017186:Titrimetry
D008715:Methionine; D008780:Methyltransferases; C022725:5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase