Rapid hydrolysis of ATP by mitochondrial F1-ATPase correlates with the filling of the second of three catalytic sites

Abstract

Strong positive catalytic cooperativity is a central feature of the binding change mechanism for F1-ATPases. However, a detail of the mechanism that remains controversial is whether the kinetic enhancement derived from using substrate-binding energy at one catalytic site to promote product release from another site occurs upon the filling of the second or third of three catalytic sites on F1. To address this question, we compare the ATP concentration dependence of the rate of ATP hydrolysis by F1 from beef heart mitochondria to the ATP concentration dependence of the level of occupancy of catalytic sites during steady-state catalysis as measured by a centrifuge filtration assay. A single Km(ATP) is observed at 77 +/- 6 microM. Analysis of the nucleotide-binding data shows that half-maximal occupancy of a second catalytic site occurs at 78 +/- 18 microM ATP. We conclude that ATP binding to a second catalytic site is sufficient to support rapid rates of catalysis.

Fig. 1.

Dependence of MF₁ catalytic-site occupancy on substrate concentration. Total and noncatalytic site binding of [³H]ANP to MF₁ were measured as described under Experimental Procedures. The stoichiometry of nucleotide bound at catalytic sites was obtained by subtracting the amount bound at noncatalytic sites from the total bound, and the results were plotted on the y axis. PP_i-treated ndMF₁ at 0.85 μM (circles) or 1.7 μM (hexagons) was incubated with varying [³H]ATP concentrations, or [³H]ATP at 50 nM (triangles) or 250 nM (inverted triangles) was incubated with varying MF₁ concentrations. Measurements were repeated omitting PP_i and using 0.85 μM (filled squares) or 1.7 μM (filled diamonds) ndMF₁.

Fig. 2.

Analysis of the binding data. The results for [³H]ANP binding to PP_i-treated ndMF₁ catalytic sites (Fig. 1, open symbols) and noncatalytic sites (filled symbols) are presented in a semilogarithmic plot. The solid line represents a best fit of the data (open symbols) to an equation N = N_m·(2[ATP]² + [ATP]·K_s,2)/([ATP]² + [ATP]·K_s,2 + K_s,1·K_s,2) that describes the sequential occupancy of two catalytic sites on MF₁, where N is the measured stoichiometry of nucleotide bound at catalytic sites, K_s,1 and K_s,2 are ATP concentrations required for half-maximal occupancy of the first and second sites, respectively, and N_m is the stoichiometry of ANP that binds to each site at saturation. The best-fit values obtained by a nonlinear regression analysis for K_s,1, K_s,2, and N_m are ≤15 nM, 78 ± 18 μM, and 1.00 ± 0.02 mol/mol of MF₁, respectively. It should be noted that, as expected from the large difference between K_s,1 and K_s,2, the same values were obtained for a fit that does not require sequential binding.

Fig. 3.

Dependence of MF₁ rates of catalysis on substrate concentration. The rate of ATP hydrolysis by PP_i-treated ndMF₁ was measured as described under Experimental Procedures. Solid line, best fit of the data to the Michaelis–Menten equation (K_m = 77 ± 6 μM; V_max = 367 ± 8 s^–1). (Inset) An Eadie–Hofstee analysis of the same data.

Fig. 4.

A model for the mechanism of ATP synthesis and hydrolysis by F_oF₁-ATP synthases. Each schematic cross section of F₁, as viewed from the membrane surface, depicts three stationary catalytic sites (different shades of blue/green) surrounding the asymmetric rotor shaft of the γ-subunit (yellow). The orientation of γ determines the properties of the catalytic sites, where the conformational states are defined as follows: C, a high-affinity catalytic site at which ATP forms reversibly and spontaneously [designated in earlier versions of this scheme (54) as T for tight]; D, a conformation of the catalytic site that binds ADP preferentially; D′, a conformation that readily binds both ADP and P_i; T, a conformation that binds ATP preferentially; and O, a low-affinity transitional conformation between the D and T states. In the upper pathway, ATP synthesis at the site positioned at 4 o'clock occurs by sequential passage through intermediate stages 1 to 5. The clockwise (cw) rotational steps (1 → 2 and 3 → 4) are driven by proton transport through F_o. In the lower pathway, ATP hydrolysis at the site positioned at 12 o'clock occurs by sequential passage through intermediate stages 1 to 5′. In this case, the counterclockwise (ccw) rotational steps are driven by ATP binding energy derived from the T → C transition (2′ → 3′) and by the D′ → D conformational change (4′ → 5′). Only one third of the catalytic cycle is shown for either synthesis or hydrolysis. In the complete cycle, γ rotates 360°. Form 1 is identical to forms 5 and 5′ except that γ has rotated +120° and –120°, respectively, with the associated binding changes.