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.
School of Chemistry, University of Sydney, Sydney, New South Wales, Australia.
Anne
A
Pilotelle-Bunner
Flemming
F
Cornelius
Pierre
P
Sebban
Philip W
PW
Kuchel
Ronald J
RJ
Clarke
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