title: Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri.
abstract: Enzymological studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted in order to identify and characterize physiologically relevant substrates and reactions in acetyl-CoA synthesis and decomposition in methanogens. Whereas previous investigations employed carbon monoxide as substrate and reducing agent for acetyl-CoA synthesis, we discovered that bicarbonate (or CO2) acts as a highly efficient carbonyl group precursor substrate in the presence of either hydrogen or Ti3+.EDTA as reducing agent. In reactions with Ti3+.EDTA, synthesis of acetyl-CoA was strongly dependent on ferredoxin, and in reactions with H2, dependence on ferredoxin was absolute. Two major hydrogenases were resolved from the enzyme complex preparation by HPLC gel filtration. One of these hydrogenases was shown to be active in reconstitution of acetyl-CoA synthesis in CO2-containing reactions with H2 as reducing agent. The hydrogenase active in reconstitution was capable of reducing ferredoxin, but was unreactive toward the 8-hydroxy-5-deazaflavin derivative coenzyme F420. In contrast, the hydrogenase that did not reconstitute acetyl-CoA synthesis was reactive with F420 but was unable to reduce ferredoxin. Further experiments were performed in which the value of the equilibrium constant (Keq) was determined for the reaction: H2 + CO2 + CH3-H4SPt + CoASH <--> acetyl-CoA + H4SPt + H2O, where CH3-H4SPt and H4SPt stand for N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. Keq for this reaction was found to be 2.09 x 10(6) M-1ATMH2-1 at 37 degrees C. Calculations of thermodynamic values for additional, related reactions were made and are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Uniformed Services University of the Health Sciences, Department of Biochemistry, Bethesda, Maryland 20814, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Grahame
authors.forename: E
authors.initials: E
authors.lastname: DeMoll
mesh_terms: D000105:Acetyl Coenzyme A / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D017021:Methanosarcina barkeri / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D011506:Proteins; D000105:Acetyl Coenzyme A