Identification of the residues determining the substrate specificities of the two human mitochondrial ornithine carrier isoforms

The mitochondrial carrier family consists of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has suggested that these transporters have a common substrate binding site consisting of three major contact points in the central carrier cavity. Here we have probed the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 with site-directed mutagenesis and a set of different substrates in transport assays. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Together with further mutations we demonstrate that the residues in position 179 and E180 of contact point II selects the stereospecificity and side chain length of the amino acid substrates, suggesting that these residues bind the substrate carboxylate and alpha-amino group, respectively. Residue E77 of contact point I probably interacts with the terminal amino group of the substrate side chain. For the substrate-induced conformational changes required for substrate translocation, it is likely that all three contact points are involved by R179 connecting to R275 of contact point III with cation-pi interactions through W224. Mutations at position 179 also severely affect the turnover number, implying that substrate binding to residue 179 is a rate-limiting step in the catalytic transport cycle. Since R179 is located in vicinity of the matrix gate of the cavity, it may be a key residue in the opening of the carrier to the matrix side.