An acyl-coenzyme a chain length dependent assay for 3-oxoacyl-coenzyme a thiolases employing acetyldithio-coenzyme A

✍ Scribed by Louise V. Wrensford; Chris Coppola; Vernon E. Abderson

• Publisher: Elsevier Science
• Year: 1991
• Tongue: English
• Weight: 640 KB
• Volume: 192
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(91)90181-r

✦ Synopsis

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.