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

P. E. Jablonski, J. G. Ferry

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)2481-2487
Number of pages7
JournalJournal of bacteriology
Volume173
Issue number8
DOIs
StatePublished - 1991

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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