ABSTRACT
3-Hydroxy-3-methyl-1-thionoglutaryl-coenzyme A, a dithioester analog of 3-hydroxy-3-methylglutaryl-CoA, has been enzymatically synthesized using the HMG-CoA synthase catalyzed condensation of acetyl-CoA with 3-oxo-1-thionobutyryl-CoA. HMGdithio-CoA is a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase. Inhibition was mainly competitive with respect to HMG-CoA with a Kis of 0.086 +/- .01 microM and noncompetitive with respect to NADH with a Kis of 3.7 +/- 1.5 microM and a Kii of 0.65 +/- .05 microM in the presence of 110 microM (R.S)-HMG-CoA.
Subject(s)
Acyl Coenzyme A/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Pseudomonas/enzymology , Acyl Coenzyme A/chemical synthesis , Animals , Chickens , Chromatography, DEAE-Cellulose , Chromatography, Ion Exchange , Hydroxymethylglutaryl-CoA Synthase/isolation & purification , Hydroxymethylglutaryl-CoA Synthase/metabolism , Indicators and Reagents , Kinetics , Liver/enzymology , Protein Binding , UltrafiltrationABSTRACT
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.
