Dual mechanisms of allosteric acceleration of the Na(+),K(+)-ATPase by ATP

Abstract

Investigations of the E2 --> E1 conformational change of Na(+),K(+)-ATPase from shark rectal gland and pig kidney via the stopped-flow technique have revealed major differences in the kinetics and mechanisms of the two enzymes. Mammalian kidney Na(+),K(+)-ATPase appears to exist in a diprotomeric (alphabeta)(2) state in the absence of ATP, with protein-protein interactions between the alpha-subunits causing an inhibition of the transition, which occurs as a two-step process: E2:E2 --> E2:E1 --> E1:E1. This is evidenced by a biphasicity in the observed kinetics. Binding of ATP to the E1 or E2 states causes the kinetics to become monophasic and accelerate, which can be explained by an ATP-induced dissociation of the diprotomer into separate alphabeta protomers and relief of the preexisting inhibition. In the case of enzyme from shark rectal gland, the observed kinetics are monophasic at all ATP concentrations, indicating a monoprotomeric enzyme; however, an acceleration of the E2 --> E1 transition by ATP still occurs, to a maximum rate constant of 182 (+/- 6) s(-1). This indicates that ATP has two separate mechanisms whereby it accelerates the E2 --> E1 transition of Na(+),K(+)-ATPase alphabeta protomers and (alphabeta)(2) diprotomers.

Figure 1

Stopped-flow fluorescence transients of Na⁺,K⁺-ATPase from (A) shark rectal gland and (B) pig kidney noncovalently labeled with RH421 (500 nM, after mixing). Na⁺,K⁺-ATPase (40 μg/mL or 0.27 μM, after mixing) was rapidly mixed with an equal volume of a solution containing 130 mM NaCl (pH 7.4, 24°C). Both the enzyme suspensions and the NaCl solution were prepared in a solution containing 25 mM histidine and 0.1 mM EDTA. The fluorescence of membrane-bound RH421 was measured at an excitation wavelength of 577 nm at emission wavelengths of ≥665 nm (RG665 glass cutoff filter). The calculated observed rate constants were (A) 10.6 (± 0.3) s⁻¹ for the shark enzyme and (B) 25 (± 3) s⁻¹ (35% of the total amplitude) and 0.94 (± 0.02) s⁻¹ (65%) for the pig enzyme.

Figure 2

Stopped-flow fluorescence transients of the E2 → E1 transition of Na⁺,K⁺-ATPase from shark rectal gland nonovalently labeled with RH421. The transition was induced by rapid mixing with 130 mM NaCl and varying concentrations of Na₂ATP. All experimental conditions were as described in Fig. 1. The labels 0, 2, 5, 10, and 2000 represent the ATP concentration after mixing in μM. The calculated observed rate constants were 0 ATP, 10.6 (± 0.3) s⁻¹; 2 μM ATP, 17 (± 1) s⁻¹; 5 μM ATP, 23 (± 3) s⁻¹; 10 μM ATP, 32 (± 2) s⁻¹; and 2000 μM ATP 166 (± 4) s⁻¹.

Figure 3

Dependence of the observed rate constant of the RH421 fluorescence change on the concentration of Na₂ATP (after mixing) for stopped-flow experiments in which Na⁺,K⁺-ATPase from shark rectal gland in a solution containing 25 mM histidine and 0.1 mM EDTA was rapidly mixed with the same histidine/EDTA solution containing 130 mM NaCl and varying concentrations of Na₂ATP. [Na⁺,K⁺-ATPase] = 40 μg/mL (0.27 μM), [RH421] = 500 nM; pH = 7.4, T = 24°C. The solid line is a nonlinear least-squares fit of the data to Eq. 1. The fit parameters were k_obs^min = 9 (± 6) s⁻¹; k_obs^max = 182 (± 6) s⁻¹; and K_d = 63 (± 12) μM.

Figure 4

(A) Stopped-flow fluorescence transients of Na⁺,K⁺-ATPase from pig kidney noncovalently labeled with RH421 (500 nM, after mixing). Na⁺,K⁺-ATPase (40 μg/mL or 0.27 μM, after mixing) was rapidly mixed with an equal volume of a solution containing 130 mM NaCl and varying concentrations of Na₂ATP (pH 7.4, 24°C). Both the enzyme suspension and the NaCl/Na₂ATP solution were prepared in a solution containing 25 mM histidine and 0.1 mM EDTA. Excitation and emission wavelengths were as described in the caption to Fig. 3. The calculated observed rate constants were (a) 0.5 μM ATP, 24 (± 3) s⁻¹ (31% of the total amplitude) and 1.32 (± 0.02) s⁻¹ (69%); (b) 2 μM ATP, 27 (± 4) s⁻¹ (27%) and 1.86 (± 0.03) s⁻¹ (73%); (c) 150 μM ATP 20.4 (± 0.2) s⁻¹; and (d) 1500 μM ATP 36.3 (± 0.2) s⁻¹. (B) Kinetic simulations of the experimental fluorescence transients based on a reversible dimeric conformational change model (see Fig. 5) and the rate constants, equilibrium constants, and fluorescence levels given in Table 1.

Figure 5

Amplitudes (ΔF/F₀) of the fluorescence change observed in stopped-flow measurements on pig kidney Na⁺,K⁺-ATPase under the conditions described in Fig. 3 as a function of the ATP concentration. ΔF is the total fluorescence change (i.e., of both phases if a two-phase signal was observed), and F₀ is the initial fluorescence level before mixing with NaCl and ATP. The solid line represents the prediction of simulations based on a reversible dimeric conformational model (see Fig. 6). The values of ΔF/F₀ were all determined from extrapolations to infinite time of either single or double exponential fits (whichever were more appropriate on the basis of residual plots) to the kinetic data.

Figure 6

Reversible dimeric model of the E2 → E1 conformational change of pig kidney Na⁺,K⁺-ATPase.