Escherichia coli coenzyme A-transferase: Kinetics,catalytic pathway and structure

The inducible acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli catalyzes the transfer of CoA from acetyl-CoA to acetoacetate by a mechanism involving a covalent enzyme-CoA compound as a reaction intermediate. Acetyl-CoA + enzyme ? enzyme-CoA + Acetate Enzyme-CoA + acetoacetate ? acetoacetyl-CoA + enzyme These conclusions are based on the following data: 1) In the absence of acetoacetate, the maximal velocity of exchange of [¹⁴C]acetate into acetyl-CoA was comparable with maximal velocity of the complete reaction. 2) Incubation of the enzyme with NaBH₄ after preincubation with an acyl-CoA substrate inactivated the enzyme by reduction of a glutamate residue in the β subunit of the CoA-transferase to α-amino-δ-hydroxyvaleric acid. Given the susceptibility of thioesters to borohydride reduction, the enzyme-CoA bond is a γ-glutamyl thiolester 3) Following incubation of the enzyme with a fluorescent derivative of acetyl-CoA, 1,N⁶-ethenoacetyl-CoA, etheno-CoA was bound to the CoA-transferase. Free etheno-CoA did not bind to the enzyme.