Rb+ occlusion in dephosphorylation intermediates of the gastric H,K‑ATPase catalytic cycle

SANTIAGO E. FARAJ; MÓNICA R. MONTES; WANDA M. VALSECCHI; ROLANDO C. ROSSI; NICOLE T. CERF

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofisica

Rb tight binding during E2P dephosphorylationSanti, Wanda, Nicole, Rolando, MMThe enzyme catalyses H+ transport at the expense of ATP hydrolysis through a mechanism traditionally described by the E1-E2 reaction cycle. In the E1 conformation, H+ are bound to the cytoplasmic side of the pump, leading to pump phosphorylation and the formation of the E1P intermediate, which is converted to the E2P conformation with the concomitant release of H+ at the opposite side of the membrane. K+ acces from the lumen induces the E2P dephosphorylation and then K+ is trapped within the membrane domain in what is called the occluded form. A transition to E1 faces the ion sites to the cytoplasmic side and the cycle termina when K+ is released into the cell. As it is proposed for the P-type ATPases, the sequence of physiological dephosphorylation reaction consists in a sequence leading from the E2-P ground state to the E2.Pi product state that occlude K+. Although Na+/K+- ATPase and H+/K+-ATPase are very similar in their amino-acidic sequence and kinetic mechanisms, it has not been possible in the gastric pump the isolation of the state holding one occluded K+ per enzyme unit. It has been shown that vanadate promotes Rb ( a K congener) occlusion (,) and that vanadate and fluorinated complexes (..,..) induce structures that represent different intermediates in the E2-P→E2 dephosphorylation sequence. In this work we analyzed the relationship between these intermediates and its ability to bind and occlude Rb+. Results show that the level of Rb occlusion increases from the E2P product state to the E2P ground state in accordance with shift from a luminal-closely to a luminal-open conformation.