Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II)

The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine, Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II), Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-ol-der kinetics, The reversal of the inhibition by hydroxylamine and the pK(a) (7.1) of the modified residue indicated that the residue was a histidine, The order of the inactivation kinetics showed that 1 mol of histidine was modified per mol of CPT-II.

When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity, The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely, It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated CT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced, Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine.