ATP Synthesis:- ATP Is Stabilized Relative to ADP on the Surface of F1
Isotope exchange experiments with purified F1 reveal a remarkable fact about the enzyme’s catalytic mechanism: on the enzyme surface, the reaction ADP+Pi⇌ ATP+H2O is readily reversible—the free-energy change for ATP synthesis is close to zero! When ATP is hydrolyzed by F1 in the presence of 18O-labeled water, the Pi released contains an 18O atom. Careful measurement of the 18O content of Pi formed in vitro by F1 catalyzed hydrolysis of ATP reveals that the Pi has not one, but three or four 18O atoms (Fig. 19–21). This indicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before Pi leaves the enzyme surface. With Pi free to tumble in its binding site, each hydrolysis inserts 18O randomly at one of the four positions in the molecule. This exchange reaction occurs in unenergized FoF1complexes (with no proton gradient) and with isolated F1—the exchange does not require the input of energy. Kinetic studies of the initial rates of ATP synthesis and hydrolysis confirm the conclusion that ΔG0 for ATP synthesis on the enzyme is near zero. From the measured rates of hydrolysis (k1=10 s-1) and synthesis (k-1=24 s-1), the calculated equilibrium constant for the reaction
Enz-ATP ⇌Enz–(ADP+Pi) Is
From this Keq, the calculated apparent ΔG is close to zero. This is much different from the Keq of about 105 (ΔG=-30.5kJ/mol) for the hydrolysis of ATP free in solution (not on the enzyme surface).
What accounts for the huge difference? ATP synthase stabilizes ATP relative to ADP+Pi by binding ATP more tightly, releasing enough energy to counterbalance the cost of making ATP. Careful measurements of the binding constants show that FoF1binds ATP with very high affinity (Kd≤10-12M) and ADP with much lower affinity (Kd≈10-5M). The difference in Kd corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward formation of the product ATP.
FIGURE 19–21 Catalytic mechanism of F1. (a) 18O-exchange experiment. F1 solubilized from mitochondrial membranes is incubated with ATP in the presence of 18O-labeled water. At intervals, a sample of the solution is withdrawn and analyzed for the incorporation of 18O into the Pi produced from ATP hydrolysis. In minutes, the Pi contains three or four 18O atoms, indicating that both ATP hydrolysis and ATP synthesis have occurred several times during the incubation. (b) The likely transition state complex for ATP hydrolysis and synthesis in ATP synthase (derived from PDB ID 1BMF). The α subunit is shown in green, in gray. The positively charged residues β-Arg182 and α -Arg376 coordinate two oxygens of the pentavalent phosphate intermediate; Lys155 interacts with a third oxygen, and the Mg2+ ion (green sphere) further stabilizes the intermediate. The blue sphere represents the leaving group (H2O). These interactions result in the ready equilibration of ATP and ADP Pi in the active site.
