N super(5)-methyltetra-hydromethanopterin:coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N super(5)-methyltetra-hydromethanopterin (CH sub(3)-H sub(4)MPT) to coenzyme M (H-S-CoM) in methanogenic archaebacteria. With method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri) and in H sub(2)/CO sub(2)-grown Methanobacterium thermoautotrophicum) were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH sub(3)-S-CoM formation from CH sub(3)-H sub(4)MPT (apparent K sub(m) = 50 mu M) and H-S-CoM (apparent K sub(m) = 250 mu M) of approximately 0.6 mu mol/min/mg protein. For activity the presence of Ti(III) citrate (apparent K sub(m) = 15 mu M) and of ATP (apparent K sub(m) = 30 mu M) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH sub(3)-H sub(4)MPT in the absence of H-S-CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH sub(3)-H sub(4)MPT to H-S-CoM in the absence of ATP and Ti(III). Demethylation in the presence of H-S-CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.