Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.
The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.
Abbanat
D R
DR
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Ferry
J G
JG
D000085:Acetates / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology; D019605:Euryarchaeota / Q000378:metabolism*; D006847:Hydrocarbons, Iodinated / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000085:Acetates; D003486:Cyanides; D006847:Hydrocarbons, Iodinated; D009097:Multienzyme Complexes; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D002248:Carbon Monoxide; C014055:methyl iodide; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase