Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-L-carnitine.

Abstract

Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[(14)C]bromoacetyl-l-carnitine yields N-3-carboxy[(14)C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue.