title: An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A.
abstract: 1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.
mesh_terms: D000105:Acetyl Coenzyme A; D000214:Acyl Coenzyme A; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002848:Chromatography, DEAE-Cellulose; D002854:Chromatography, Paper; D003065:Coenzyme A; D004355:Drug Stability; D005977:Glutarates; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D005977:Glutarates; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D000105:Acetyl Coenzyme A; D007652:Oxo-Acid-Lyases; D003065:Coenzyme A