ATP Synthesis:- Rotational Catalysis Is Key to the Binding-Change Mechanism for ATP Synthesis
On the basis of detailed kinetic and binding studies of the reactions catalyzed by FoF1, Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis (Fig. 19–24). A given β sub unit starts in the β-ADP con formation, which binds ADP and Pi from the surrounding medium. The subunit now changes conformation, assuming the -ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of ADP+Pi with ATP on the enzyme surface. Finally, the subunit changes to the β-empty conformation, which has very low affinity for ATP, and the newly synthesized ATP leaves the en zyme surface. Another round of catalysis begins when this subunit again assumes the β-ADP form and binds ADP and Pi.
The conformational changes central to this mechanism are driven by the passage of protons through the Fo portion of ATP synthase. The streaming of protons through the Fo “pore” causes the cylinder of c subunits and the attached subunit to rotate about the long axis of γ, which is perpendicular to the plane of the mem brane. The subunit passes through the center of the α3β3 spheroid, which is held stationary relative to the membrane surface by the b2 and δ subunits (Fig. 19–23f). With each rotation of 120, comes into con tact with a different subunit, and the contact forces that subunit into the β-empty conformation. The three subunits interact in such a way that when one assumes the β-empty conformation, its neighbor to one side must assume the β-ADP form, and the other neighbor the β-ATP form. Thus, one complete rotation of the γ subunit causes each β subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface.
One strong prediction of this binding-change model is that the γ subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr. The rotation of γ in a single F1 molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to γ and watching it move relative to α3β3 im mobilized on a microscope slide, as ATP was hydrolyzed. When the entire FoF1 complex (not just F1) was used in a similar experiment, the entire ring of c subunits rotated with (Fig. 19–25). The “shaft” rotated in the predicted direction through 360. The rotation was not smooth, but occurred in three discrete steps of 120o. As calculated from the known rate of ATP hydrolysis by one F1 molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in con verting chemical energy into motion is close to 100%. It is, in Boyer’s words, “a splendid molecular machine!”
FIGURE 19–24 Binding-change model for ATP synthase. The F1 com plex has three nonequivalent adenine nucleotide–binding sites, one for each pair of α and β subunits. At any given moment, one of these sites is in the β-ATP conformation (which binds ATP tightly), a second is in the β-ADP (loose-binding) conformation, and a third is in the β- empty (very-loose-binding) conformation. The proton-motive force causes rotation of the central shaft—the γ subunit, shown as a green arrowhead—which comes into contact with each αβ subunit pair in succession. This produces a cooperative conformational change in which the β-ATP site is converted to the -empty conformation, and ATP dissociates; the β-ADP site is converted to the β-ATP conformation, which promotes condensation of bound ADP Pi to form ATP; and the -empty site becomes a β -ADP site, which loosely binds ADP+Pi entering from the solvent. This model, based on experimental findings, requires that at least two of the three catalytic sites alternate in activity; ATP cannot be released from one site unless and until ADP and Pi are bound at the other.
