The Biosynthesis of β-Hydroxy-β-methylglutaryl Coenzyme A in Yeast

Abstract

Abstract Attempts to separate β-hydroxy-β-methylglutaric acid coenzyme A-condensing enzyme and thiolase from yeast preparations by density gradient centrifugation, chromatography, and electrophoresis are described. In each case, it was not possible to prepare by these methods condensing enzyme free of thiolase activity. The results suggest that both proteins have closely similar physical properties. Each enzyme can be inactivated independently of the other by treatment with various reagents. Trypsin and chymotrypsin partially inactivate condensing enzyme. Papain, on the other hand, had no effect. The prior presence of acetoacetyl coenzyme A protects the condensing enzyme from inactivation by trypsin and chymotrypsin. Thiolase is completely inactivated by trypsin and chymotrypsin. The presence of CoA prior to treatment with trypsin partially protects thiolase from inactivation. Papain inactivates thiolase about 50% in the absence of substrates, but in the presence of acetoacetyl-CoA the inactivation goes rapidly to completion. Heat treatment inhibits condensing enzyme but not thiolase. The presence of acetyl-CoA protects condensing enzyme from heat inactivation and at the same time makes thiolase somewhat sensitive to heat. In agreement with previous results, iodoacetamide completely inhibits thiolase and only partially affects condensing enzyme. The presence of acetoacetyl-CoA or acetyl-CoA prior to the addition of iodoacetamide gives almost complete protection to condensing enzyme and partial protection to thiolase. CoA did not protect thiolase from inhibition. These results concerning the effect of substrates suggest that conformational changes in both condensing enzyme and thiolase are induced by the substrates, acetyl-CoA, acetoacetyl-CoA, and CoA. Kinetic measurements on condensing enzyme show that acetoacetyl-CoA exerts a large inhibitory effect. At low concentrations of acetyl-CoA, acetoacetyl-CoA appears to act as a competitive inhibitor. In the case of thiolase both acetoacetyl-CoA and CoA show potent substrate inhibition. Specificity studies with various acyl thioesters indicate that the thiolase activity studied differs from the β-ketothiolase and mixed thiolase studied by Stern and Drummond (4, 18).