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Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase
P L Jorgensen1, J M Nielsen, J H Rasmussen
1Biomembrane Research Center, August Krogh Institute, University of Copenhagen, Denmark. PLJorgensen@AKI.KU.DK
Acta Physiologica Scandinavica. Supplementum
|October 28, 1998
Summary
This study reveals key residues in Na,K-ATPase mutations essential for ATP binding and cation occlusion. Specific carboxylate groups are critical for high-affinity potassium ion binding, impacting enzyme function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Kinetics
Background:
- Na,K-ATPase (sodium-potassium adenosine triphosphatase) is a vital ion pump.
- Understanding its function is crucial for cellular physiology and disease research.
- Mutations can disrupt ion binding and enzyme activity.
Purpose of the Study:
- To investigate ATP and cation binding in Na,K-ATPase mutations.
- To identify residues involved in ion occlusion and E1-E2 transitions.
- To elucidate the role of specific carboxylate groups in ion binding affinity.
Main Methods:
- Expression of Na,K-ATPase mutations in yeast cells.
- Equilibrium binding measurements of ATP and cations (T1+).
- Analysis of enzyme kinetics and affinity (Kd).
Main Results:
- Identified Arg546 as potentially involved in ATP binding.
- Wild-type enzyme occludes two T1+ ions with high affinity.
- Mutations in Glu327, Asp804, Asp808, and Glu779 significantly reduced or abolished T1+ ion occlusion and affinity.
- Substitution of Glu779 reduced occlusion to one T1+ ion and decreased affinity.
Conclusions:
- Specific carboxylate groups in transmembrane segments 4, 5, and 6 are essential for high-affinity occlusion of potassium ions.
- These residues play a critical role in Na,K-ATPase function and ion transport.
- The findings provide insights into the mechanism of ion binding and enzyme transitions.