N-Benzoyl-D-isoleucinol

The mammalian glucose transporter, GLUT1, is capable of alternating between two conformations which expose either an outward- or inward-facing ligand binding site. The possibility that these conformational changes are related to the presence of prolines and glycines in transmembrane region 10 was investigated by site-directed mutagenesis. Chinese hamster ovary clones which were transfected with Pro385-->Ile and Pro385-->glycine mutations of GLUT1 were shown, by Western blotting and cell surface carbohydrate labelling, to have expression levels which were comparable with the wild type. The transport activity was markedly reduced as a result of the Pro385-->isoleucine but not in the Pro385-->glycine mutation. The loss of transport activity in the Pro385-->isoleucine clone was associated with loss of labeling by the exofacial photoaffinity ligand, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA), but there was no loss in labeling by the inside site-directed ligand cytochalasin B. These results suggest that the transporter cannot adopt the outward-directed conformation in the Pro385-->isoleucine clone. By contrast, the glycine substitution for proline at this position resulted in a retention of the ligand binding properties at both inside and outside sites. We suggest a putative mode of operation of the transporter which involves conformational flexibility about the prolines in transmembrane segment 10 such that helices 11 and 12 can alternately either pack against the outside (ATB-BMPA binding) site in helices 7, 8, and 9 or against the inner (cytochalasin B binding) site at the base of transmembrane segment 10.

Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.