3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.
Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.
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D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003065:Coenzyme A / Q000494:pharmacology; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046911:Macromolecular Substances; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D013696:Temperature
D046911:Macromolecular Substances; D011773:Pyruvates; C014674:methyl methanethiosulfonate; C017092:bromopyruvate; D000105:Acetyl Coenzyme A; C033104:2-bromoacetyl coenzyme A; D008741:Methyl Methanesulfonate; D006904:Hydroxymethylglutaryl-CoA Synthase; D007652:Oxo-Acid-Lyases; D003065:Coenzyme A