A Research Journey with ATP Synthase

dicyclohexylcarbodiimide These reflections present a perspective of how I and my graduate students and postdoctoral fellows, over a span of many years, arrived at the concept that ATP is made by an unusual rotational catalysis of the ATP synthase. A recent sketch of the structure of this remarkable enzyme is given in Fig. 1. Such a depiction is the culmination of the efforts of many investigators. 1Except for a few instances, the mention of important advances in information about the ATP synthase and in related areas of biochemistry is included without specific references. The objective of this contribution is not to provide a review of the field and to recognize priorities for contributions but to note how various advances impacted on studies by my group. 1Except for a few instances, the mention of important advances in information about the ATP synthase and in related areas of biochemistry is included without specific references. The objective of this contribution is not to provide a review of the field and to recognize priorities for contributions but to note how various advances impacted on studies by my group. The two portions of the enzyme are the membrane-imbedded F0 and the attached F1 that has three catalytic sites, principally on the large β subunits. ATP is formed when protons pass through the F0, driving the rotation of the ring-shaped cluster ofc subunits and the attached ε and γ subunits. Other subunits attached to outer portions of the F0 and F1 served as a stator. The internal rotary movement of the γ subunit is coupled to sequential changes in the conformation of the catalytic sites. During ATP synthesis these conformational changes promote the binding of ADP and Pi, the formation of tightly bound ATP, and the release of ATP. Revealing the mechanism of the ATP synthase became a major research goal in the latter part of my long career. This paper recalls how my career developed as related to the remarkable progress in biochemical knowledge. It presents the background and results of fruitful, as well as mistaken, approaches that were explored. Born and educated through college in Utah, at the age of 21 I entered graduate school in the Department of Biochemistry at the University of Wisconsin in the fall of 1939. The biochemical research and teaching there were excellent. Not until years later did I appreciate all that is necessary to create such a fine scientific environment. I had had no previous courses or research experience in biochemistry and was uncertain about my career choice. By the end of my first year of graduate study the fascination of biochemical understanding and the addictive effect of experimental attempts to uncover new knowledge had firmly launched me toward a career in biochemical research. The Department of Biochemistry at Wisconsin was at the forefront of research in nutrition and metabolism. Recent achievements included the identification of nicotinic acid as a vitamin, the irradiation of milk to produce vitamin D, the discovery of a vitamin K antagonist (dicoumarin), and the discovery of lipoic acid as a growth factor for bacteria. At that time incoming graduate students were assigned to a mentor professor. Both Henry Lardy, from South Dakota, and I joined the group of Professor Paul Phillips whose major interest was in dairy cattle nutrition. Evidence had been obtained that vitamin C might help prevent reproductive difficulties in cattle, and one of my assignments was to find if vitamin C might ameliorate the reproductive failure that occurred in rats with vitamin E deficiency. No benefits of vitamin C were noted, but the rats also showed the striking muscular dystrophy characteristic of vitamin E deficiency. Exploration, together with Henry Lardy, of the possible cause of this dystrophy led me into study of ATP-related enzymes. Henry is still active in an exceptionally distinguished career that has included major contributions to the understanding of oxidative phosphorylation. The milieu at Wisconsin (meetings where students and staff discussed recent research papers, frequent research seminars, and class instructions) introduced me to the wonder of enzyme catalysis. A prominent event was a symposium on respiratory enzymes at which the outstanding biochemists Meyerhof, Cori, Ochoa, Lipmann, Kalckar and others contributed (1Symposium on Respiratory Enzymes (1942) University of Wisconsin Press, Madison, WI.Google Scholar). From this and other sources I learned that ATP and phosphorylations were central to the capture and use of energy derived from foodstuffs. Perhaps defective formation of ATP might underlie the muscle dystrophy in my vitamin E-deficient rats. One approach was to measure the ability of muscle extracts to make phosphocreatine during glycolysis. No definitive defect from vitamin E deficiency was found, but in the course of these experiments, I noted a stimulation of the transfer of phosphate from 3-phosphoglycerate to creatine by K+ ions. This was traced to a requirement of K+ for transfer of the phosphoryl group from 2-phosphoenolpyruvate to ADP. The discovery of the K+ activation of pyruvate kinase was the first demonstration of a K+ requirement for an enzyme reaction. The two Journal of Biological Chemistry publications reporting this were the best of several from my graduate studies (2Boyer P.D. Henry A. Phillips P.H. J. Biol. Chem. 1942; 146: 673-682Abstract Full Text PDF Google Scholar,3Boyer P.D. Lardy H.A. Phillips P.H. J. Biol. Chem. 1943; 149: 529-541Abstract Full Text PDF Google Scholar). An understanding of the K+ activation was attained at the University of Wisconsin some 50 years later from the x-ray structure of pyruvate kinase (4Larsen T.M. Benning M.M. Rayment I. Reed G.H. Biochemistry. 1998; 37: 6247-6255Crossref PubMed Scopus (117) Google Scholar). The K+, coordinated to four protein ligands, to an oxygen of the γ-phosphate of ATP, and to a water oxygen, apparently provides a requisite positive charge. Oxidative phosphorylation was discovered only 7 years before I started graduate studies. As noted in an interesting Prefatory chapter by Englehardt in Annual Reviews of Biochemistry (5Engelhardt W.A. Annu. Rev. Biochem. 1982; 51: 1-7Crossref PubMed Scopus (20) Google Scholar), ATP was discovered by Lohmann in 1927, and oxidative phosphorylation was first demonstrated by Engelhardt and Liubimova in 1932. These salient contributions at that time seemed far from recent to me, and discoveries such as that of cell-free fermentation by Buchner made about 40 years earlier were relegated to the distant past—science after the escape from the Middle Ages. Now, from my present perspective, research of 30 years ago still seems fairly recent and vibrant. Time seems to go much faster, but it is I who has changed while a unit of time has retained its constant value.