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.

The local electric field within phospholipid membranes modulates the charge transfer reactions in reaction centres.

Three different cholesterol derivatives and phloretin, known to affect the local electric field in phospholipid membranes, have been introduced into Rhodobacter sphaeroides reaction centre-containing phospholipid liposomes. We show that cholesterol and 6-ketocholestanol significantly slow down the interquinone first electron transfer (approximately 10 times), whereas phloretin and 5-cholesten-3beta-ol-7-one leave the kinetics essentially unchanged. Interestingly, the two former compounds have been shown to increase the dipole potential, whereas the two latter decrease it. We also measured in isolated RCs the rates of the electron and proton transfers at the first flash. Over the pH range 7-10.5 both reactions display biphasic behaviors with nearly superimposable rates and amplitudes, suggesting that the gating process limiting the first electron transfer is indeed the coupled proton entry. We therefore interpret the effects of cholesterol and 6-ketocholestanol as due to dipole concentration producing an increased free energy barrier for protons to enter the protein perpendicular to the membrane. We also report for the first time in R. sphaeroides RCs, at room temperature, a biphasicity of the P(+)Q(A)(-) charge recombination, induced by the presence of cholesterol derivatives in proteoliposomes. We propose that these molecules decrease the equilibration time between two RC conformations, therefore revealing their presence.

ATP binding equilibria of the Na(+),K(+)-ATPase.

Reported values of the dissociation constant, K(d), of ATP with the E1 conformation of the Na(+),K(+)-ATPase fall in two distinct ranges depending on how it is measured. Equilibrium binding studies yield values of 0.1-0.6 microM, whereas presteady-state kinetic studies yield values of 3-14 microM. It is unacceptable that K(d) varies with the experimental method of its determination. Using simulations of the expected equilibrium behavior for different binding models based on thermodynamic data obtained from isothermal titration calorimetry we show that this apparent discrepancy can be explained in part by the presence in presteady-state kinetic studies of excess Mg(2+) ions, which compete with the enzyme for the available ATP. Another important contributing factor is an inaccurate assumption in the majority of presteady-state kinetic studies of a rapid relaxation of the ATP binding reaction on the time scale of the subsequent phosphorylation. However, these two factors alone are insufficient to explain the previously observed presteady-state kinetic behavior. In addition one must assume that there are two E1-ATP binding equilibria. Because crystal structures of P-type ATPases indicate only a single bound ATP per alpha-subunit, the only explanation consistent with both crystal structural and kinetic data is that the enzyme exists as an (alphabeta)(2) diprotomer, with protein-protein interactions between adjacent alpha-subunits producing two ATP affinities. We propose that in equilibrium measurements the measured K(d) is due to binding of ATP to one alpha-subunit, whereas in presteady-state kinetic studies, the measured apparent K(d) is due to the binding of ATP to both alpha-subunits within the diprotomer.