Substrate stereochemistry of the acetyl-CoA acetyltransferase reaction.
1. A specimen of symmetrically tritiated 3S-3-hydroxy[2-3H2]butyryl-CoA was prepared from acetoacetyl-CoA by incubation in tritiated water, followed by enzymic reduction using 3S-specific 3-hydroxyacyl-CoA dehydrogenase. In addition, a specimen of dideuterated, racemic 3RS-3-hydroxy-[2-2H2]butyryl-CoA was prepared chemically from 3RS-3-hydroxy[2-2H2]butyric acid. 2. These acyl-CoA derivatives were incubated respectively with deuterium oxide and tritated water in presence of enoyl-CoA hydratase. Stereospecifically tritiated compounds, respectively 2S,3S-3-hydroxy[2-2H1,3H1]butyryl-CoA and 2R,3S-3-hydroxy[2-1H1,3H1]butyryl-CoA (plus 3R-diastereomer) were formed and isolated. 3. 3S-3-Hydroxy[2-3H2]butyryl-CoA and the 3S-[2-3H2,3-14C]-labelled material were also prepared as described in the preceding paper. The latter substrate was used to establish conditions in which little loss of tritium would occur (found; about 7%) on cleavage to acetyl-CoA in the presence of 3-hydroxyacyl-CoA dehydrogenase and acetyl-CoA acetyltransferase. Little loss of tritium indicates a small degree of racemization of the stereospecifically 2-tritiated 3-hydroxyacyl-CoA derivatives at the level of transiently formed acetoacetyl-CoA and acetyl-CoA. This ensures high optical purity of the chiral acetates generated from the stereospecifically tritiated hydroxybutyryl-CoA specimens listed in paragraph (2). 4. These two acyl-CoA derivatives and the symmetrically tritiated 3S-3-hydroxy[2-3H2]butyryl-CoA listed in paragraph (3) were converted to the acetates enzymically in the sequence hydroxy-butyryl-CoA leads to acetoacetyl-CoA leads to acetyl-CoA leads to acetate. The isolated acetates were assayed for chirality by conversion to the malates and fumarates as usual. 5. The malate formed from 3S-3-hydroxy[2-3H2]butyryl-CoA on incubation with fumarase lost nearly 50%, that derived from 2R,3S-3-hydroxy[2-2H1,3H1]butyryl-CoA (plus 3R-diastereomer) retained about 26% and that derived from 2S,3S-3-hydroxy[2-1H1,3H1]butyryl-CoA retained about 78% of its total tritium content. 6. It was concluded that the detachment of acetyl-CoA from acetoacetyl-CoA on acetyl-CoA acetyltransferase occurs with inversion of configuration at the methylene group which becomes methyl.
D000090:Acetoacetates; D000101:Acetyl-CoA C-Acetyltransferase; D000123:Acetyltransferases; D000429:Alcohol Oxidoreductases; D003065:Coenzyme A / Q000138:chemical synthesis; D003437:Crotonates; D003903:Deuterium; D005649:Fumarate Hydratase; D006880:Hydroxy Acids; D006885:Hydroxybutyrates / Q000138:chemical synthesis; D007553:Isotope Labeling; D007700:Kinetics; D008025:Ligases; D008956:Models, Chemical; D013237:Stereoisomerism; D014316:Tritium
D000090:Acetoacetates; D003437:Crotonates; D006880:Hydroxy Acids; D006885:Hydroxybutyrates; D014316:Tritium; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase; D005649:Fumarate Hydratase; D008025:Ligases; D003065:Coenzyme A