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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. Ulver full discography torrent. 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.

Introduction An important role of Mg 2+ in biology is as a cofactor of ATP. The Mg 2+ ion is complexed by the negatively charged oxygens of its phosphate groups. The Mg 2+ is thus thought to help shield the negative charges of the phosphates, allowing reaction with the electron pairs of attacking groups and facilitating phosphoryl transfer (). One of the most important enzymes in which this is the case is the Na +,K +-ATPase, which is responsible for maintaining electrochemical potential gradients for Na + and K + across the plasma membrane. To our knowledge, no crystal structure of the Na +,K +-ATPase in the E1 state with bound Mg 2+ and ATP has yet been reported. Nevertheless, based on a published crystal structure of the related enzyme sarcoplasmic reticulum Ca 2+-ATPase (), and using computer modeling, Patchornik et al.

() suggested that, like the phosphates of ATP, the aspartate residues D710, D443, and D714 contribute to Mg 2+ coordination. Although this may be correct, it is difficult from crystal structural data to make conclusions about the relative strengths of interactions.

The aim of this article is to provide reliable experimental data on the strength of binding of Mg 2+ ions to the Na +,K +-ATPase. A difficulty in studying Mg 2+ interaction with the Na +,K +-ATPase under physiological conditions, i.e., in the presence of ATP and Na + ions, is that it immediately induces phosphorylation, so that Mg 2+ binding cannot be separated from the phosphorylation reaction. This precludes equilibrium binding studies. Here we have, therefore, applied a pre-steady-state kinetic technique (stopped-flow spectrofluorimetry) utilizing the voltage-sensitive fluorescent probe RH421. Together with data recently obtained under the same experimental conditions for Mg 2+ binding to ATP (), this allows us to analyze the question of the relative contributions of the enzyme and ATP in binding Mg 2+ in the enzyme-ATP-Mg 2+ complex.

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In the case of the E1 conformation of the enzyme, it will be shown here that, although the enzyme environment is definitely important for the catalysis of phosphoryl transfer from ATP, coordination by ATP dominates the dissociation constant ( K d) for Mg 2+ binding. Enzyme and reagents Na +,K +-ATPase-containing membrane fragments from shark rectal glands were purified essentially as described by Skou and Esmann (). The specific ATPase activity at 37°C and pH 7.4 was measured according to the methods of Ottolenghi (). The activity of the preparation used was 1679 μmol ATP hydrolyzed h −1 (mg of protein) −1 and the protein concentration was 4.82 mg mL −1. The protein concentration was determined according to the Peterson modification () of the Lowry method () using bovine serum albumin as a standard.