Professor, Chemical Resources Laboratory, Tokyo Institute of Technology
Mystery behind bioenergy
—Proving the rotation of ATP synthase—
“Living organisms do not favor rotation.” It was surprising that Prof. Yoshida who has proved the rotation of ATP (adenosine triphosphate) synthase said that. “There are no living organisms with wheels. Isn’t that interesting?” says Prof. Yoshida.
ATP is a small molecule with the molecular weight of over 500. When ATP turns into ADP (adenosine diphosphate) and inorganic phosphate (Pi), it generates large energy of 7.5kcal per mole. Nearly all the bioenergy is supplied by the ATP hydrolysis. However, there are only 10 to 20g of ATP in the human body. It cannot be conserved in the body; therefore, the human body produces and consumes roughly the same amount of ATP as his or her own weight in a day.
ATP plays such an important role for vital activity, and yet the research on ATP synthase had not progressed. When Prof. Paul Boyer advocated the rotation of ATP synthase 20 years ago, nobody listened to him. It was 1994 when things turned around. Dr. John Walker clarified the three-dimensional structure of ATP synthase. When Prof. Yoshida saw the stalk at the center of a spherical hexamer, he was shocked by the possibility of its rotation because he had not believed the ATP synthase would rotate. And then, he decided to do the experiments to prove the ATP synthase surely rotates.
It was difficult to prove it because the ATP synthase was too small to observe with an optical microscope. So, Prof. Yoshida fixed the only 10nm-diameter hexamer surrounding the stalk with fluorescently labeled 1µm-length actin filaments on the coverslip, and succeeded in visualization and videotaping the rotation of ATP synthase. “Everyone was surprised including myself because the primitive enzyme in every living organism rotated. It was believed that living organisms did not favor rotation.” From the experiment, he has found out that ATP synthase rotates the actin filaments at a high speed of 8 revolutions per second. According to his calculation, it rotates with nearly perfect energy efficiency.
The 1997 Nobel Prize went to Prof. Jens Skou for his discovery of an ion-transporting enzyme and Prof. Paul Boyer and Dr. John Walker for elucidation of the enzymatic mechanism underlying the synthesis of ATP. The image of the rotation of ATP synthase was played in the preliminary review board and it made their receiving the Nobel Prizes definite. Prof. Yoshida was thought to share the Nobel Prize, unfortunately, he missed receiving. He said, “The Nobel Prize is for the persons who proposed a very original idea, persisted and prevailed. Boyer deserved it.”
What Prof. Yoshida is currently concerned is that it seems fewer researchers conduct high-risk researches. “They have to write papers continually to get research funds, so they intend to conduct research with a future. However, it is also very important to work on research even under uncertainty because it may turn out novel achievement.” says Prof. Yoshida. He expects those courageous researchers will open new era with their bold ideas.
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| Fig. 2 Rotational Catalysis |
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| Prof. Boyer conducted research mainly on kinetics of F1-ATPase and came to a hypothesis on the mechanism of ATP synthesis as follows: 1) The synthesis of enzyme-bound ATP (ADP + Pi → ATP) requires no energy; 2) the energy created by proton flow is used when ADP and Pi bind to the enzyme and when ATP is released from the enzyme; 3) three catalytic sites on the enzyme alternatively participate in the reaction. He worked up a theory as Alternate Binding Change Mechanism. According to the theory, an ATP bound to one of the catalytic sites promotes the reaction of the ATP (ATP → ADP + Pi) already bound to the adjacent catalytic site. And then, the three catalytic sites go through three states of loose binding site for ATP, tight binding site for ATP, and loose binding site for ADP and Pi. Proton motive force for ATP synthesis is used for the alternate binding change of the catalytic sites.
In the Alternate Binding Change Mechanism, the three catalytic sites separately play own roles at a moment, but they actually play all the same roles over a period of time. Therefore, it was assumed that a subunit consisting of γ, δ and ε would rotate in the enzyme complexes to alternate roles equally (it turned out later that δ did not rotate, but the essence of his proposal remained valid). And the rotation was considered to transfer energy between F0 and F1. This concept is referred as Rotational Catalysis. |
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| Fig. 3 Experiment of Profs. Noji and Yasuda |
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| Although Prof. Boyer concluded rotational catalysis by distinct intuition and logical prospect, conventional biochemical research was not progressed enough to prove rotation. In 1997, Prof. Yoshida and his colleagues succeeded in the direct observation of the rotation of the γ subunit by using F1-ATPase α3β3γ subcomplex derived from thermophile as shown in the figure. |
