EFFECTS OF SUBSTRATE-BINDING AND PH ON THE SECONDARY STRUCTURE OF CARNITINE ACETYLTRANSFERASE

Carnitine acetyltransferase (CAT) exists as a monomer in solution as d emonstrated by dynamic light scattering measurements. Under these cond itions, interactions between CAT and its substrates, L-carnitine and a cetyl-CoA, were studied by circular dichroism (CD) and fluorescence sp ectroscopy over a wide range of substrate concentrations. CD data indi cated that the binding of L-carnitine and acetyl-CoA caused changes in the secondary structure of the protein. Quenching of the intrinsic pr otein fluorescence upon binding of either substrate corroborated these findings. Analysis of the binding data suggests that binding of both substrates to CAT is specific and saturable, and that there is a singl e binding site (or multiple identical and independent binding sites) a n CAT for each substrate. Estimated L-carnitine/CAT dissociation const ants were 506 +/- 58 mu M and 236 +/- 27 mu M in the absence or presen ce of acetyl-CoA, respectively. The dissociation constant for acetyl-C oA/CAT was estimated at 19 +/- 7 mu M The effect of pH on the secondar y structure of the protein was determined in order to investigate the structural cause for the pH-dependent enzymatic activity of CAT. Loss of alpha-helices and a reduction of thermal stability in CAT was detec ted at both acidic and basic pH. Thus, the reduced catalytic activity of CAT at acidic or basic pH may be due to pH-induced protein unfoldin g.