The purification and some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from Baker's yeast.
1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (mumol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5mum, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10-30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.
D000215:Acylation; D000478:Alkylation; D002729:Chloromercuribenzoates; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003065:Coenzyme A; D005033:Ethylmaleimide; D008190:Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D008722:Methods; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013439:Sulfhydryl Reagents
D002729:Chloromercuribenzoates; D013439:Sulfhydryl Reagents; D008190:Lyases; D005033:Ethylmaleimide; D003065:Coenzyme A