Purification and properties of acetyl coenzyme A synthetase from bakers' yeast.
Acetyl-CoA synthetase, utilized in a coupled reaction system, has been shown to be applicable to the spectrophotometric determination of propionic and methylmalonic acids in biological fluids. The isolation of acetyl-CoA synthetase from yeast is simpler than the purification from mammalian sources. This study also presents some properties of the yeast enzyme and compares it to the more extensively studied enzyme isolated from ammmalian tissue. Isolation and purification yielded a preparation with a specific activity of 44 units/mg at 25 degrees. The purified acetyl-CoA synthetase was apparently homogeneous by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis with an estimated subunit molecular weight of 78,000. Polyacrylamide gel electrophoresis in the presence of ATP revealed a single protein band which contained all of the enzyme activity. Analytical ultra-centrifuge studies indicated the presence of a single protein with a molecular wright of 151,000 and sedimentation velocity analysis revealed a single peak with a sedimentation coefficient of 8.65 So20,w. Similar to the enzyme from mammalian sources, yeast acetyl-CoA synthetase has a high degree of substrate specificity and is active only on acetate and propionate. In addition, the reaction mechanism, as demonstrated by initial velocity patterns obtained from substrate pairs, appeared to be identical to the enzyme from bovine heart. However, the apparent Michaelis constants for the substrates were significantly different from the mammalian enzyme. The yeast-derived enzyme also differed from the mammalian in terms of molecular weight, amino acid composition, pH optimum, effect of monovalent cations, and stability characteristics. Thus, yeast acetyl-CoA synthetase is more easily purified than the mammalian enzyme and provides an excellent preparation for the assay of propionic and methylmalonic acids.
D000106:Acetate-CoA Ligase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003066:Coenzyme A Ligases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity
D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase