High-Pressure Stopped-Flow Studies to Characterize Transient Conformational Transitions of a Membrane Enzyme: Na, K-ATPase

Abstract

The kinetics of ATP-induced phosphorylation and K⁺ binding of Na,K-ATPase has been investigated at different pressures by applying the fluorescence stopped-flow method. For both type of reactions, a conformational transition is considered to act as rate-limiting reaction step. These transitions are characterized by large activation volumes up to +100 ml mol⁻¹. The significance of the determined values in terms of Kramers’ theory is discussed. A molecular interpretation related to solvation changes and cavity formation in the transmembrane domain of the protein is suggested. A large negative reaction volume is found upon ATP binding (—100 ml mol⁻¹). The selective binding of Na⁺ and K⁺ leads to smaller, but positive values.