Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: an inhibition kinetic approach combined with the analysis of the homology structural model.

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism.

Chemical modification of the mitochondrial ornithine/citrulline carrier by SH reagents: effects on the transport activity and transition from carrier to pore-like function.

The transport activity of the purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria was correlated to modification of its sulfhydryl groups by various reagents. Both the ornithine/ornithine (antiport) and the ornithine/H(+) (unidirectional) transport modes catalysed by the ornithine/citrulline carrier were inhibited by methanethiosulfonates, mercurial reagents, N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The treatment of the ornithine/citrulline carrier with mercurial reagents, at concentrations above 5 microM, caused the induction of an additional (pore-like) transport mode, characterized by loss of substrate specificity and a transport activity higher than that of the unmodified carrier.