Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.
Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.
D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives* / Q000378:metabolism; D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D000214:Acyl Coenzyme A; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003600:Cytosol / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D000214:Acyl Coenzyme A; C010667:acetoacetyl CoA; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase