How to digest heat denaturation: differential stability of gastric H,K-ATPase cycle intermediates

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

Jornada; Virtual Activity YIB-Talks; 2020

The Young Initiative on Biophysics (IUPAB-SAB)

The gastric H,K-ATPase is an integral membrane protein responsible for gastric acid secretion. The enzyme couple the energy released from ATP hydrolysis to the transport of H+ 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, while the counter-transport ion, K+, binds from the lumen to the E2 conformation.Protein-ligand interactions play a key role not only in determining the structure and function of a protein, but also in its stability. Since cation binding shifts the E1−E2 conformational equilibrium and considering that E1 states of Na,K ATPase are more thermolabile than E2 forms1, we examined if different conformational states of the H,K ATPase differ in their stability. Firstly, we measured the kinetics of enzyme activity decay at several temperatures in the range of 45 to 55 °C, finding that the H,K ATPase is more labile than the Na,K ATPase. Then, to examine the possible dependence of thermolability with the conformational states of the enzyme, we analyzed the variations induce by K+ and Na+?which has shown to shift the equilibrium toward the E1 conformation2?in activity and conformational experiments. In both cases, we observed that K+ reduces the velocity of inactivation, whereas the kinetics is less affected by Na+. These results suggest that the E2 conformation is significantly more stable than E1; however, contrastingly with the Na,K ATPase, this feature cannot be explained by the existence of a K+-occluded state since it was not favored in the experimental conditions2.