title: An acyl-coenzyme A chain length dependent assay for 3-oxoacyl-coenzyme A thiolases employing acetyldithio-coenzyme A.
abstract: An assay for 3-oxoacyl-coenzyme A (3-oxoacyl-CoA) thiolases is described. The reaction utilizes acetyldithio-CoA as the nucleophile and variable chain length saturated acyl-CoA's as the electrophiles. The properties of the 3-oxoacyl-CoA dithioester product, notably a pKa of 6.6 +/- 0.1 and an extinction coefficient of 21,600 cm-1 M-1 for the enethiolate at 357 nm, make it possible to spectrophotometrically follow the reaction in the thermodynamically unfavorable carbon-carbon bond-forming direction. These properties eliminate both the background decomposition and the dependence on Mg2+, chain length, and pH that complicate assays with 3-oxoacyl-CoA substrates. Purified thiolase I from pig liver was 140-fold more active with butyryl-CoA as the electrophile than with acetyl-CoA and 38-fold more reactive with hexanoyl-CoA than with myristoyl-CoA. Beef liver homogenate showed a much greater relative activity with myristoyl-CoA as the electrophile than either purified pig heart thiolase I or pig heart homogenate. The analysis of the separation of thiolases by anion-exchange chromatography is simplified and further suggests the existence of isozymes with varying chain length specificities.
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912.
authors.forename: L V
authors.initials: LV
authors.lastname: Wrensford
authors.forename: C
authors.initials: C
authors.lastname: Coppola
authors.forename: V E
authors.initials: VE
authors.lastname: Anderson
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000214:Acyl Coenzyme A; C059703:acetyldithio-coenzyme A; D000102:Acetyl-CoA C-Acyltransferase