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 [1-14C]acetyl-CoA yielded protein-bound acetyl groups. The Keq. for the acetylation was 1.2 at pH7.0 and 4°C. Acetyl-labelled synthase was isolated free from [1-14C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-14C]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 [1-14C]acetyl-CoA or 3-hydroxy-3-methyl-[14C]glutaryl-CoA respectively. 4. The isolated [1-14C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4°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.
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Research Article| January 01 1974
An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A
B. Middleton ;
Biochem J (1974) 137 (1): 15–23.
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B. Middleton, P. K. Tubbs; An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A. Biochem J 1 January 1974; 137 (1): 15–23. doi: https://doi.org/10.1042/bj1370015
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