8885ac19690-7503/1/0411:32 AM Page709 6mac76:385 19.2 ATP Synthes FIGURE 19-22 Reaction coordinate ATP (in solution) ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction(left), reaching the transition state () between substrate and ADP+P oduct is the major energy barrier to overcome E·ATP In the reaction catalyzed by ATP synthase(right), elease of ATP from the enzyme, not formation of E·ADP+P ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and Pi n aqueous solution is large and positive, but on E+S provides sufficient binding energy to6.。 the enzyme surface, the very tight binding of ATP energy of the enzyme-bound atP close to that of ADP Pi so the reaction is readily reversible Reaction coordinate ATP synthase The equilibrium constant is near 1. The free Typical enzy energy required for the release of ATP is provided by the proton-motive force four positions in the molecule. This exchange reaction It is the proton gradient that causes the enzyme to re- occurs in unenergized Fo Fi complexes(with no proton lease the ATP formed on its surface. The reaction Co- gradient) and with isolated Fl-the exchange does not ordinate diagram of the process(Fig. 19-22)illustrates require the input of energy the difference between the mechanism of ATP synthase Kinetic studies of the initial rates of ATP synthesis and that of many other enzymes that catalyze ender and hydrolysis confirm the conclusion that AG for atP gonic reactions synthesis on the enzyme is near zero. From the meas- For the continued synthesis of ATP, the enzyme ured rates of hydrolysis (,= 10s)and synthesis must cycle between a form that binds ATP very tightly Ck-1=24s), the calculated equilibrium constant for and a form that releases ATP. Chemical and crystallo- the reaction graphic studies of the ATP synthase have revealed the structural basis for this alternation in function Enz.ATP Enz-(ADP P Each B Subunit of ATP Synthase Can Assume Three Different Conformations 2.4 Mitochondrial FI has nine subunits of five different this Keo, the calculated apparent AG"o is close to types, with the composition a3B3yoe. Each of the three This is much different from the k of about 10 B subunits has one catalytic site for ATP synthesis. The (AG"=-30.5 kJ/mol) for the hydrolysis of ATP free in crystallographic determination of the F, structure by solution(not on the enzyme surface) John E. Walker and colleagues revealed structural de- What accounts for the huge difference? ATP syn tails very helpful in explaining the catalytic mechanism thase stabilizes ATP relative to ADP + Pi by binding ATP of the enzyme. The knoblike portion of Fi is a flattened more tightly, releasing enough energy to counterbalance sphere, 8 nm high and 10 nm across, consisting of al- the cost of making ATP. Careful measurements of the erating a and B subunits arranged like the sections of binding constants show that FoFl binds ATP with ver an orange(Fig. 19-23a-c). The polypeptides that make high affinity(Ka s 10-2 M) and ADP with much lower up the stalk in the Fi crystal structure are asymmetri cally arranged, with one domain of the single y subunit to a difference of about 40 kJ/mol in binding energy, and making up a central shaft that passes through Fi, and his binding energy drives the equilibrium toward for mation of the product ATP. the three B subunits, designated B-empty(Fig. 19-23c) Although the amino acid sequences of the three B sub- The Proton gradient Drives the release of atp units are identical, their conformations differ, in part from the Enzyme Surface because of the association of the y subunit with just one of the three. The structures of the s and e subunits are Although ATP synthase equilibrates ATP with ADP not revealed in these crystallographic studies Pi, in the absence of a proton gradient the newly syn- The conformational differences among B subunits thesized atP does not leave the surface of the enzyme. extend to differences in their ATP/ADP-binding sitesfour positions in the molecule. This exchange reaction occurs in unenergized FoF1 complexes (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 G for ATP synthesis on the enzyme is near zero. From the meas￾ured rates of hydrolysis (k1  10 s1 ) and synthesis (k1  24 s1 ), the calculated equilibrium constant for the reaction Enz-ATP Enz–(ADP
Pi) is Keq   2.4 From this Keq , the calculated apparent G is close to zero. This is much different from the Keq of about 105 (G  30.5 kJ/mol) for the hydrolysis of ATP free in solution (not on the enzyme surface). What accounts for the huge difference? ATP syn￾thase 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 FoF1 binds ATP with very high affinity (Kd ≤ 1012 M) and ADP with much lower affinity (Kd ≈ 105 M). The difference in Kd corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward for￾mation of the product ATP. The Proton Gradient Drives the Release of ATP from the Enzyme Surface Although ATP synthase equilibrates ATP with ADP
Pi , in the absence of a proton gradient the newly syn￾thesized ATP does not leave the surface of the enzyme. 24 s 1 10 s1 k1 k1 yz It is the proton gradient that causes the enzyme to re￾lease the ATP formed on its surface. The reaction co￾ordinate diagram of the process (Fig. 19–22) illustrates the difference between the mechanism of ATP synthase and that of many other enzymes that catalyze ender￾gonic reactions. For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. Chemical and crystallo￾graphic studies of the ATP synthase have revealed the structural basis for this alternation in function. Each
Subunit of ATP Synthase Can Assume Three Different Conformations Mitochondrial F1 has nine subunits of five different types, with the composition 3
3. Each of the three
subunits has one catalytic site for ATP synthesis. The crystallographic determination of the F1 structure by John E. Walker and colleagues revealed structural de￾tails very helpful in explaining the catalytic mechanism of the enzyme. The knoblike portion of F1 is a flattened sphere, 8 nm high and 10 nm across, consisting of al￾ternating and
subunits arranged like the sections of an orange (Fig. 19–23a–c). The polypeptides that make up the stalk in the F1 crystal structure are asymmetri￾cally arranged, with one domain of the single subunit making up a central shaft that passes through F1, and another domain of associated primarily with one of the three
subunits, designated
-empty (Fig. 19–23c). Although the amino acid sequences of the three
sub￾units are identical, their conformations differ, in part because of the association of the subunit with just one of the three. The structures of the  and  subunits are not revealed in these crystallographic studies. The conformational differences among
subunits extend to differences in their ATP/ADP-binding sites. 19.2 ATP Synthesis 709 G (kJ/mol) Reaction coordinate 80 60 40 20 0 ‡ P ADP
Pi ES E
S E ADP
Pi [E ATP] Typical enzyme ATP synthase ATP (in solution) FIGURE 19–22 Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (left), reaching the transition state (‡) between substrate and product is the major energy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from the enzyme, not formation of ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and Pi in aqueous solution is large and positive, but on the enzyme surface, the very tight binding of ATP provides sufficient binding energy to bring the free energy of the enzyme-bound ATP close to that of ADP
Pi, so the reaction is readily reversible. The equilibrium constant is near 1. The free energy required for the release of ATP is provided by the proton-motive force. 8885d_c19_690-750 3/1/04 11:32 AM Page 709 mac76 mac76:385_reb: