The molecular weight and thiol residues of acetyl-coenzyme A synthetase from ox heart mitochondria.
1. A constant molecular weight of 57000 was obtained by gel filtration of highly purified acetyl-CoA synthetase over a 1000-fold range of enzyme concentrations. The amino acid analysis is reported. 2. With native enzyme at 20 degrees C the relatively rapid reaction of four thiol residues with p-hydroxymercuribenzoate caused an immediate inhibition reversible by either CoA or mercaptoethanol. Other substrates did not protect against this rapid inhibition. 3. The much slower reaction of the remaining four thiol residues was independent of the concentration of the mercurial, first-order with respect to enzyme, and had a large energy of activation (+136kJ/mol), suggesting that a conformation change in the protein was rate-limiting. This slow phase of the reaction was accompanied by an irreversible inactivation of the enzyme. 4. The effects of substrates on this irreversible inactivation at pH7.0 in 5 mm-MgCl(2) indicated strong binding of ATP and pyrophosphate by the enzyme (concentrations for half-maximal effects, K((1/2)), were <30mum and <10mum respectively) and weaker binding of acetyl-CoA (K((1/2)) about 1 mm), AMP (K((1/2)) about 2mm) and acetate. In the presence of acetate, MgCl(2) and p-hydroxymercuribenzoate, titration of the enzyme with ATP revealed at least two ATP binding sites/mol. 5. The experiments suggest that reaction of the thiol residues with mercurial causes loss of enzymic activity by altering the structure of the enzyme, rather than that the thiol residues play a direct role in the catalysis.
D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003066:Coenzyme A Ligases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000032:analysis*; D004589:Electrophoresis, Disc; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates; D007700:Kinetics; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014461:Ultracentrifugation
D000596:Amino Acids; D006902:Hydroxymercuribenzoates; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D003545:Cysteine