The energetics and sodium-ion dependence of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase studied with cob(I)alamin as methyl acceptor and methylcob(III)alamin as methyl donor.
N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3-H4MPT) to an enzyme-bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H-S-CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3-H4MPT and cob(I)alamin (reaction a), and methyl-coenzyme M formation from methylcob(III)alamin and H-S-CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3-H4MPT to H-S-CoM. The free energy changes (delta G0') associated with reactions a and b were both between -10 kJ/mol and -20 kJ/mol, consistent with a free energy change of approximately -30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium-ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme-bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H-S-CoM is a site of coupling with sodium-ion translocation.
Weiss
D S
DS
Max-Planck-Institut für terrestrische Mikrobiologie, Philipps-Universität, Marburg, Germany.
Gärtner
P
P
Thauer
R K
RK
D004734:Energy Metabolism; D017014:Methanobacterium / Q000201:enzymology; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology*; D013816:Thermodynamics; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000378:metabolism*
D011622:Pterins; C073313:5-methyl-5,6,7,8-tetrahydro-methanopterin; D012964:Sodium; C019476:mecobalamin; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase; D014805:Vitamin B 12