Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A.
3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue.
Miziorko
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Behnke
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D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000494:pharmacology*; D000818:Animals; D002250:Carbon Radioisotopes; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors*; D010761:Phosphorus Radioisotopes
D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; C046467:3-chloropropionyl-coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase; D007652:Oxo-Acid-Lyases