Weiss D S, Gärtner P, Thauer R K
Max-Planck-Institut für terrestrische Mikrobiologie, Philipps-Universität, Marburg, Germany.
Eur J Biochem. 1994 Dec 15;226(3):799-809. doi: 10.1111/j.1432-1033.1994.00799.x.
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.
来自产甲烷古菌的N5-甲基四氢甲蝶呤：辅酶M甲基转移酶是一种与膜相关的酶复合物，它利用甲基转移反应驱动一个能保存能量的钠离子泵。甲基转移分两步进行，首先从N5-甲基四氢甲蝶呤（CH3-H4MPT）转移到与酶结合的钴胺素（I）辅基上，其次从甲基化的钴胺素转移到辅酶M（H-S-CoM）上。在本研究中，我们报道甲基转移酶还能分别使用外源钴胺素（I）和甲基钴胺素（III）作为甲基受体和甲基供体。该酶催化由CH3-H4MPT和钴胺素（I）形成甲基钴胺素（III）（反应a），以及由甲基钴胺素（III）和H-S-CoM形成甲基辅酶M（反应b）。这两个反应均显示是可逆的。反应a的催化速率约为（3 U/mg），反应b的催化速率约为生理反应（即从CH3-H4MPT到H-S-CoM的甲基转移）速率的10%（0.3 U/mg）。与反应a和b相关的自由能变化（ΔG0'）均在-10 kJ/mol至-20 kJ/mol之间，与生理反应确定的约-30 kJ/mol的自由能变化一致。反应b而非反应a依赖钠离子。假设外源钴胺素（I）的甲基化和外源甲基钴胺素（III）的去甲基化模拟了与酶结合的类咕啉辅基的甲基化和去甲基化，可以推断从甲基化的钴胺素辅基到H-S-CoM的甲基转移是与钠离子转运偶联的位点。
