Properties of the two isoenzymes of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum.
Methyl-coenzyme M reductase (MCR) catalyses the methane-forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl-coenzyme M (CH3-S-CoM) by 7-mercaptoheptanoylthreonine phosphate (H-S-HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial-velocity measurements of the two-substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary-complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H-S-HTP and for CH3-S-CoM and in Vmax. MCR I displayed a Km for H-S-HTP of 0.1-0.3 mM, a Km for CH3-S-CoM of 0.6-0.8 mM and a Vmax of about 6 mumol.min-1 x mg-1 (most active preparation). MCR II showed a Km for H-S-HTP of 0.4-0.6 mM, a Km for CH3-S-CoM of 1.3-1.5 mM and a Vmax of about 21 mumol.min-1 x mg-1 (most active preparation). The pH optimum of MCR I was 7.0-7.5 and that of MCR II 7.5-8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existence of MCR isoenzymes in M. thermoautotrophicum is discussed.
Bonacker
L G
LG
Max-Planck-Institut für Terrestrische Mikrobiologie Marburg, Germany.
Baudner
S
S
Mörschel
E
E
Böcher
R
R
Thauer
R K
RK
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015080:Mesna / Q000031:analogs & derivatives / Q000378:metabolism; D008697:Methane / Q000378:metabolism*; D017014:Methanobacterium / Q000201:enzymology* / Q000254:growth & development; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; C023349:methyl coenzyme M; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna; D008697:Methane