Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila

Methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila TM-1 was purified 16-fold from a cell extract to apparent homogeneity as determined by native polyacrylamide gel electrophoresis. Ninety-four percent of the methylreductase activity was recovered in the soluble fraction of cell extracts. The estimated native molecular weight of the enzyme was between 132,000 (standard deviation [SD], 1,200) and 141,000 (SD, 1,200). Denaturing polyacrylamide gel electrophoresis revealed three protein bands corresponding to molecular weights of 69,000 (SD, 1,200), 42,000 (SD, 1,200), and 33,000 (SD, 1,200) and indicated a subunit configuration of α₁β₁γ₁. As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin. ATP stimulated enzyme reactivation and was postulated to be involved in a conformational change of the inactive enzyme from an unready state to a ready state that could be reductively reactivated. The temperature and pH optima for enzyme activity were 60°C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F₄₃₀ per mol of enzyme (M(r), 144,000). The K(m)s for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 μM, respectively.