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TITLE: Kinetics of Substrate Interaction with the Sodium Pump

SPEAKER: Professor Promod R. Pratap,

TIME: Thursday Oct. 2, 2003 at 4 PM

PLACE: George P. Williams, Jr. Lecture Hall, (Olin 101)

The sodium pump (Na⁺/K⁺-ATPase) is a membrane-associated protein that uses energy from the hydrolysis of ATP to move 3 Na⁺ out of and 2 K⁺ into the cell. This protein accounts for approximately 25% of the energy usage of a body at rest. The pump converts chemical energy stored in ATP into electrochemical potential energy stored in ion gradients across the cell membrane. In addition, this pump is the target for digitalis and other drugs that belong to the class of cardiac glycosides. Our lab is interested in the kinetics and thermodynamics of the sodium pump. We would like to characterize how the pump utilizes energy to perform its function, and to determine the energetic cost for each step in the pump reaction cycle. Our approach in this regard is to determine the rate-constants for each step in the reaction cycle of the pump using equilibrium and transient kinetic techniques. From these rate-constants, we then determine the free energy changes associated with each step. In this talk, I will focus on conformational changes in the pump induced when ATP binds to the pump. We have examined these changes using Fourier transform infrared (FTIR) spectroscopy, under both equilibrium and transient conditions. For transient kinetic experiments, we initiated ATP binding to the pump by releasing ATP from an inert caged precursor, and followed the conformational change with time. For equilibrium kinetic experiments, we used attenuated total internal reflection (ATR) FTIR spectros copy to measure conformational changes under varying ATP concentrations. From these experiments: The standard free energy change for ATP-induced conformational change in the presence of sodium, sodium + potassium, or magnesium was estimated to be +10 kJ/mol; The half-maximal ATP concentration for the conformational change measured with FTIR was an order of magnitude higher in the absence of cations that bind to the protein, indicating that this conformational change occurs more spontaneously in the absence of cations than in their presence. I will conclude this talk with a discussion of the implications of these observations.