Substrate stereochemistry of the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans.
The steric course of the decarboxylation of glutaconyl-CoA to crotonyl-CoA, catalysed by the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans, was elucidated using the sequence: chiral acetate----citrate----glutamate----glutaconyl-CoA----crotonyl-CoA ----chiral acetate. Since glutaconyl-CoA or glutaconate labeled at C-4 was subjected to rapid chemical or enzymatic exchanges, glutamate was fermented to acetate by growing cells of A. fermentans. The analysis of the final chiral acetates gave following deviations from 50% in the fumarase exchange: + 13.8% starting with (R)-acetate and - 13.9% starting with (S)-acetate. The results demonstrated a retention of configuration during the decarboxylation. Thus glutaconyl-CoA decarboxylase adds to the list of biotin enzymes in which exclusive retention of configuration was observed. Glutaconate CoA-transferase from A. fermentans catalysed a 3H exchange of [2,4,4-3H]glutaconate with water when acetyl-CoA was present. At low concentration of acetyl-CoA (20 microM) the exchange ceased after exactly one atom 3H was released into the water, at high concentrations (1 mM) the exchange proceeded further. The apparent Km of acetyl-CoA in the exchange (1.1 microM) was 150 times smaller than that of the complete CoA transfer. It was concluded that either a mixed anhydride, between a carboxyl group of the enzyme and [2,4,4-3H]glutaconate, or enzyme-bound glutaconyl-CoA was the exchanging species.
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D000085:Acetates / Q000096:biosynthesis; D001710:Biotin / Q000494:pharmacology*; D002262:Carboxy-Lyases / Q000378:metabolism*; D002384:Catalysis; D005285:Fermentation; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity; D045854:Veillonellaceae / Q000201:enzymology*
D000085:Acetates; D007473:Ion Channels; D001710:Biotin; D012964:Sodium; D002262:Carboxy-Lyases; C023060:glutaconyl CoA decarboxylase