Conditions for the self-catalysed inactivation of carnitine acetyltransferase. A novel form of enzyme inhibition

Abstract

1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(−)-carnitine plus CoA or of bromoacetyl-CoA plus (−)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromoacetyl substrate analogue; in each case about 1mole of inhibitor is required to inactivate completely 1mole of enzyme of molecular weight 58000±3000. 3. Inhibition by bromoacetyl-CoA plus (−)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (−)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (−)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4°. 5. Incubation with S-carboxymethyl-CoA (−)-carnitine ester causes a slow inhibition of carnitine acetyltransferase. 6. The formation of bound S-carboxymethyl-CoA (−)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme–substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase.