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 alpha 1 beta 1 gamma 1. 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 degrees C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (Mr, 144,000). The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 microM, respectively.
Jablonski
P E
PE
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0305.
Ferry
J G
JG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000494:pharmacology; D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000378:metabolism*; D005288:Ferredoxins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D002951:Citrates; D005288:Ferredoxins; D009097:Multienzyme Complexes; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D019343:Citric Acid; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C023895:methyl coenzyme M methylreductase; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase