Catalytic mechanism of P-glycoprotein

We generated Chinese hamster ovary cells which are highly multidrug-resistant by selection in colchicine. Purified plasma membranes from these cells are enriched in P-glycoprotein (Pgp), up to 32% w/w of membrane protein. From plasma membranes we purified Pgp to homogeneity and reconstituted it in proteoliposomes. Both plasma membranes and purified reconstituted Pgp show drug-stimulated ATPase activity (approximately 20 s-1), comparable to other transport ATPases. These materials enable investigation and characterization of the catalytic sites and mechanism. Various approaches have been used, notably enzyme kinetics, photoaffinity and other covalent labelling, use of vanadate as transition-state analog, and inhibition by beryllium and aluminum fluoride. Both Pgp nucleotide sites hydrolyse MgATP and are of relatively low specificity and affinity for nucleotides. Trapping of nucleotide by vanadate in either site blocks catalysis at both sites; covalent inactivation of either site completely blocks turnover. Therefore the catalytic sites interact strongly, and it appears that when one site enters the transition-state conformation the other site is prohibited from doing so. A minimal reaction scheme for ATP hydrolysis has been determined. We have proposed an alternating catalytic sites scheme, in which drug-transport is coupled to relaxation of a high chemical potential conformation of the catalytic site (Pgp.MgADP.Pi) which is generated by the hydrolysis step itself. Photoaffinity labelling of Pgp catalytic sites has revealed equivalent Tyr residues which lie close to the adenine ring of bound MgATP in both sites.