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. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH(2) residues of the active site should be comparable to the distance between the gamma-NH(2) and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.
Tonazzi
Annamaria
A
National Research Council Institute of Biomembranes and Bioenergetics (IBBE), via Amendola 165/A, 70126, Bari, Italy.
Giangregorio
Nicola
N
Palmieri
Ferdinando
F
Indiveri
Cesare
C
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D026923:Amino Acid Transport Systems, Basic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002956:Citrulline / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D009952:Ornithine / Q000378:metabolism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D040681:Structural Homology, Protein; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D026923:Amino Acid Transport Systems, Basic; D013439:Sulfhydryl Reagents; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002956:Citrulline; D011732:Pyridoxal Phosphate; D009952:Ornithine; D008239:Lysine; D003545:Cysteine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid