AustralieFrV1, Main, Exploration, bibRecord, 007B94

Mechanism of Mg2+ Binding in the Na+,K+-ATPase

Identifieur interne : 007B94 ( Main/Exploration ); précédent : 007B93; suivant : 007B95

Mechanism of Mg2+ Binding in the Na+,K+-ATPase

Auteurs : Anne Pilotelle-Bunner [Australie, France] ; Flemming Cornelius [Danemark] ; Pierre Sebban [France] ; Philip W. Kuchel [Australie] ; Ronald J. Clarke [Australie]

Source :

Biophysical Journal [ 0006-3495 ] ; 2009.

RBID : PMC:2711396

Abstract

The Mg²⁺ 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 Mg²⁺ 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 Mg²⁺ 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 Mg²⁺-ATP interaction in solution. Within the enzyme-ATP-Mg²⁺ complex, the actual K_d for Mg²⁺ 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.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711396

DOI: 10.1016/j.bpj.2009.01.042
PubMed: 19413981
PubMed Central: 2711396

Affiliations:

Le document en format XML

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<front><div type="abstract" xml:lang="en"><p>The Mg<sup>2+</sup>
 dependence of the kinetics of the phosphorylation and conformational changes of Na<sup>+</sup>
,K<sup>+</sup>
-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<sup>+</sup>
)<sub>3</sub>
 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<sup>2+</sup>
 to saturating values of 195 (± 6) s<sup>−1</sup>
 and 54 (± 8) s<sup>−1</sup>
 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 (<italic>K</italic>
<sub>d</sub>
) of Mg<sup>2+</sup>
 interaction with the E1ATP(Na<sup>+</sup>
)<sub>3</sub>
 state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the <italic>K</italic>
<sub>d</sub>
 of Mg<sup>2+</sup>
-ATP interaction in solution. Within the enzyme-ATP-Mg<sup>2+</sup>
 complex, the actual <italic>K</italic>
<sub>d</sub>
 for Mg<sup>2+</sup> 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.
</p>
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Mechanism of Mg2+ Binding in the Na+,K+-ATPase

Mechanism of Mg2+ Binding in the Na+,K+-ATPase

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