Investigating the Energy Transduction Mechanism of P-type ATPases with Fe2+-Catalyzed Oxidative Cleavage
This paper discusses specific oxidative cleavage of renal Na+,K+-ATPase and gastric H+,K+-ATPase, catalyzed by bound Fe2+ or the complex ATP-Fe2+, and its implication for the energy transduction mechanism of P-type ATPases. The cleavage technique provides information on the spatial organization of the proteins in different conformations and, since ATP-Fe2+ substitutes for ATP-Mg2+ in activating ATPase activity, on Mg2+-ligating residues in different conformations. The experiments predict the existence of large movements of N, P, and A cytoplasmic domains accompanying E1-E2 and E1 P-E2P conformational transitionsopen in E1 conformations and closed in E2 conformations. These features fit well with the Ca2+-ATPase crystal structures in E1 or E2 conformations and also provide evidence on ATP and Mg2+ binding sites that is not available from the structures. By combining information from cleavage experiments with molecular modeling, based on the Ca2+-ATPase structure, features such as an N to P domain interaction in an E1. ATP-Mg2+ conformation can be inferred. The organization of the N, P, and A domains and the ATP and Mg2+ binding sites in the different conformational states appears to be essentially similar for Na+,K+-ATPase, H+,K+-ATPase, and Ca2+-ATPase. The oxidative cleavage technique may be a valuable tool to investigate long-range interactions that transduce the free energy of hydrolysis of ATP to active cation movements in P-type ATPases.
energy transduction mechanism fe2+-cleavage na+ k+-atpase atp-mg2+ sites