The relation of acyl transfer to the overall reaction of thiolase I from porcine heart.
Thiolase I (long chain 3-ketoacyl-CoA-specific) from porcine heart has been characterized kinetically. In the direction of acetoacetyl-CoA cleavage, a variety of thiols including CoASH show the same Vmax at saturating concentrations of acetoacetyl-CoA. At a constant overall velocity of acetoacetyl-CoA disappearance, one of the two acetyl groups from acetoacetyl-CoA will partition between CoASH and 2-mercaptoethanol at increasing 2-mercaptoethanol concentrations. These observations suggest rate-determining formation of an acetyl enzyme intermediate in the direction of acetoacetyl-CoA cleavage. In the direction of acetoacetyl-CoA formation from two molecules of acetyl-CoA, the Vmax of acetoacetyl-CoA formation is identical with the Vmax for an acetyl-CoA in equilibrium CoA isotope exchange reaction and the Vmax for an enzyme-catalyzed acetyl transfer reaction between acetyl-CoA and 2-mercaptoethanol. This suggests that in the direction of acetoacetyl-CoA synthesis, the acetyl transfer half-reaction is rate-limiting. The acetyl intermediate has been isolated and characterized. The equilibrium constant for acetyl enzyme formation from acetyl-CoA and free enzyme is 1 +/- 0.5 X 10(-2). The rate constant for spontaneous hydrolysis of the acetyl enzyme (2.6 X 10(-4) s-1) is a factor of 400 faster than the rate constant for acetyl-CoA hydrolysis under comparable conditions. The acetyl enzyme is thermodynamically and kinetically destabilized compared to acetyl-CoA.
D000105:Acetyl Coenzyme A; D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D013045:Species Specificity; D013552:Swine; D013869:Thiolester Hydrolases / Q000378:metabolism
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase; D013869:Thiolester Hydrolases