Mechanism of Mg2+ binding in the Na+,K+-ATPase.
The Mg(2+) dependence of the kinetics of the phosphorylation and conformational changes of Na(+),K(+)-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na(+))(3) state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg(2+) to saturating values of 195 (+/- 6) s(-1) and 54 (+/- 8) s(-1) for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (K(d)) of Mg(2+) interaction with the E1ATP(Na(+))(3) state was estimated as 0.069 (+/- 0.010) mM. This is virtually identical to the estimated value of the K(d) of Mg(2+)-ATP interaction in solution. Within the enzyme-ATP-Mg(2+) complex, the actual K(d) for Mg(2+) binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation.
Pilotelle-Bunner
Anne
A
School of Chemistry, University of Sydney, Sydney, New South Wales, Australia.
Cornelius
Flemming
F
Sebban
Pierre
P
Kuchel
Philip W
PW
Clarke
Ronald J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D003198:Computer Simulation; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D017711:Nonlinear Dynamics; D010766:Phosphorylation; D011487:Protein Conformation; D011726:Pyridinium Compounds; D012754:Sharks; D012965:Sodium Chloride / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013343:Styrenes
D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium