The Pentose Phosphate Pathway

Abstract

I received my basic training in enzymology as a graduate student in the laboratory of Professor T. R. Hogness at the University of Chicago from 1936 to 1939. Hogness had constructed a photoelectric spectrophotometer modeled after the one in Otto Warburg's laboratory in Berlin-Dahlem. I was assigned a problem on succinic dehydrogenase from beef heart, using the Warburg manometric apparatus, and did not get to use the spectrophotometer until Erwin Haas arrived from Warburg's laboratory in 1939. Haas asked me to join him in the search for an enzyme that would catalyze the reduction of cytochromec by reduced TPN (now NADP). This reaction was thought to be the missing link in the electron transport chain from substrate to oxygen and marked the beginning of my interest in what was then thought to function as a direct oxidative pathway for the metabolism of carbohydrate but is now known as the pentose phosphate pathway. After I left Chicago during the Second World War, my experience with the spectrophotometer landed me a job at the National Institute of Health (NIH) in Frederick S. Brackett's group in the Division of Industrial Hygiene. Brackett had assembled an automatic recording spectrophotometer in the basement of Building 2 that I was assigned to use to develop a method for the determination of carbon monoxide hemoglobin in the blood of Navy pilots returning from combat missions. That and a number of other war-related projects kept me occupied for the next 4 years. In 1945, after the end of the war with Japan, I was advised by the Director of the Laboratory, Dr. Paul Neal, that I was free to return to research in enzymology. I began studies on the reduction of cytochromec by the succinic dehydrogenase system, using what was now my own Beckman spectrophotometer. One day, which I consider to be a turning point in my career, Arthur Kornberg, who had been working in Building 4 on the biological role of folic acid, appeared in my laboratory. Arthur had become convinced that enzymes were the key to an understanding of intracellular biochemical processes and suggested that we work together. We began with studies on the effect of cyanide on the succinic dehydrogenase system, because cyanide was known to bind to and be a general inhibitor of enzymes containing the heme group. An exception was cytochrome c, which had been reported to be resistant to the action of cyanide. Contrary to these early reports, we found that cyanide did react with cytochrome c and in 1946 published our first paper together, in the Journal of Biological Chemistry, entitled “The Cytochrome c Cyanide Complex.” Two years later in 1948 when Arthur returned from a study leave in the laboratories of Severo Ochoa in New York and Carl Cori in St. Louis, he invited Leon Heppel and me to join him in setting up a new Section on Enzymes in the Laboratory of Physiology to be housed in Building 3, which was being completely renovated. Leon and I were about to be transferred, it having been discovered that the Industrial Hygiene Research Laboratory in Building 2 had never been officially part of the NIH but was in the Bureau of State Services, which was moving to new headquarters in Cincinnati. In the fall of 1948 while we waited for the of the renovation of laboratories in Building 3 to be completed, we all three worked in Building 2. Arthur and I, both of whose planned research projects would depend heavily on assays using the “pyridine nucleotide” coenzymes DPN and TPN, collaborated in their isolation. In those early years of American biochemistry there were no vendors that supplied these materials. We isolated them from sheep liver, using the unpublished procedure from Warburg's laboratory that Erwin Haas and I had used in Chicago. In 1948 Arthur and I possessed the world's supply of TPN, and when Warburg visited our laboratory in 1948, we were able to present the discoverer of TPN with a gift of 25 mg of that coenzyme. The new Enzyme Section in the Division of Physiology in Building 3 provided an exciting and stimulating atmosphere. Together with Herbert Tabor from the Laboratory of Pharmacology in Building 4, we organized a daily lunch hour journal club, during which we reviewed the literature on every facet of enzymology and intermediary metabolism. This was the beginning of a great history for Building 3, continuously occupied by scientists who were to make notable contributions in biomedical science. The first NIH recipients of the Paul Lewis Laboratories Award in Enzyme Chemistry, then one of the most prestigious awards in biological research, were all from Building 3: Arthur Kornberg in 1951, myself in 1952, and Earl Stadtman in 1953. Later, our Section on Enzymes became part of the new Experimental Biology and Medicine Institute, which Henry Sebrell, then the NIH Director, proposed to function as the basic research arm of the NIH. It was later renamed the National Institute of Arthritis and Metabolic Diseases, a change that had no effect on the nature of our research but resulted in increased funding by Congress. We continued to work in the laboratories in Building 3. Use of the pyridine nucleotides in enzyme assays with the Beckman spectrophotometer required knowledge of the exact extinction coefficients of the 340 nm peaks of the reduced forms. The published values for DPNH showed considerable variation, and there was scant information for TPNH. In the new laboratories in Building 3, Arthur and I designed experiments to determine the true extinction coefficients at 340 nm of both coenzymes, which proved to be identical. That work, published in 1948, made possible quantitative spectrophotometric measurements in reactions involving the pyridine nucleotides and became one of the most frequently cited papers in the biochemical literature. In the new laboratories in Building 3 Leon Heppel and I also collaborated in the purification of xanthine oxidase from milk after we found that this enzyme could reduce not only methylene blue, a reaction that I had studied in Chicago, but also cytochrome c. However, this reduction occurred only if oxygen was present, a curious observation that was quickly picked up by Fridovich and Handler, who were working at Duke University on the formation of the superoxide anion. The reduction of cytochrome c by superoxide anion became a widely used assay for this species of “active oxygen.” I also returned to the study of cytochromec reductase, which Haas and I had isolated from yeast, and accomplished the first isolation of this flavoprotein from mammalian liver. By then it had become apparent that these cytochromec reductases did not function in mitochondrial respiration but rather as components of the cytochrome P-450 system for the metabolism and detoxification of drugs and other xenobiotics. When Otto Warburg discovered TPN as the coenzyme required for the oxidation of glucose 6-phosphate to 6-phosphogluconate, the role of the other pyridine nucleotide, DPN, as the coenzyme required for the fermentation of glucose to ethanol in yeast or the glycolysis of glucose to lactic acid in muscle had been well established. The finding that the new coenzyme was required for the oxidation of glucose 6-phosphate and also for the further oxidation of the product, 6-phosphogluconate, led Warburg and also Frank Dickens in England and Fritz Lipmann, then working in Denmark, to propose that there existed an alternate pathway that functioned as a “direct oxidative pathway.” They had obtained evidence that the products formed in the oxidation of 6-phosphogluconate by TPN were carbon dioxide and an unidentified pentose phosphate. Because carbon dioxide was one of the products, it seemed reasonable to regard this alternate pathway as the one responsible for the oxidation of carbohydrate. Haas and I had already shown that TPN could serve as an electron transport link to the cytochromes and therefore to molecular oxygen. Twenty years after the pioneering work of Warburg, Dickens, and Lipmann, I began studies (with a new laboratory technician, Pauline (Polly) Smyrniotis) on the enzymes involved in the oxidation of 6-phosphogluconate and the metabolic intermediates formed in this pathway. We were joined by J. E. Seegmiller, my first postdoctoral student, and he and I worked out a new method for the preparation of glucose 6-phosphate and 6-phosphogluconate, which were not yet commercially available. We purified the enzyme, 6-phosphogluconate dehydrogenase, from brewers' yeast, the richest source we could find, and by coupling the reduction of TPN to its re-oxidation by pyruvate in the presence of lactic dehydrogenase showed that the first product of the oxidation of 6-phosphogloconate, in addition to carbon dioxide, was a new pentose ester, ribulose 5-phosphate, which was then converted to ribose 5-phosphate by a pentose-phosphate isomerase present in our purified dehydrogenase preparations. The separation of ribulose phosphate from ribose phosphate and the demonstration that their interconversion was catalyzed by a pentose-phosphate isomerase were made possible by the recent development at the Oak Ridge National Laboratory of a separation technique for nucleotides called ion-exchange chromatography. The identification of the sugar in the new pentose ester as ribulose was based on a number of criteria, including comparison with the authentic sugar, prepared by the method of Glatthaar and Reichstein, using a number of chemical and physical criteria, which included x-ray diffraction of the crystalline nitrophenyl hydrazones. In those days discoveries of new sugar phosphate esters were rare events, and I felt that it was necessary to establish its identity beyond a shadow of doubt. The results were first presented at the American Chemical Society meeting in Boston in 1951 at a symposium honoring Arthur Kornberg's Paul Lewis Laboratories Award, and I recall the warm reaction at that meeting to the work reported from our laboratories in Building 3. During the following year Jay Seegmiller and I showed that the same products were formed in the metabolism of 6-phosphogluconate by enzymes from mammalian tissues. An important clue to the further steps in what was later to become known as the “pentose phosphate pathway” was already in the literature. In 1938 Zaccharias Dische had demonstrated that red cell lysates catalyzed the conversion of the 5-carbon sugar, ribose 5-phosphate to hexose monophosphate, an observation that Seegmiller and I confirmed in 1952 with rabbit bone marrow extracts. These observations gave rise to the hypothesis that the oxidative pathway was really a cyclic mechanism for the direct oxidation of carbohydrate. With each turn of the cycle one molecule of carbon dioxide would be produced, and the pentose phosphates formed would be metabolized back to hexose phosphates to start another cycle. Six turns of the cycle would result in the complete oxidation of one molecule of glucose. However, the reactions involved in the conversion of the 5-carbon pentose phosphates to the 6-carbon hexose phosphates were completely unknown. What ensued was a race involving a number of laboratories, including ours at the NIH and those of Ephraim Racker at the New York City Research Laboratories, later at Cornell University in Ithaca, Seymour Cohen at the University of Pennsylvania in Philadelphia, Oliver Lampen at Washington University in St. Louis, and Frank Dickens in England, to identify the reactions and metabolic intermediates involved. It had already been established from the work of Dische and others that one of the products of pentose phosphate metabolism was the 3-carbon sugar, glyceraldehyde 3-phosphate, which suggested cleavage of a 5-carbon sugar, probably ribulose 5-phosphate, between carbon atoms 2 and 3. The 3-carbon fragment, glyceraldehyde 3-phosphate, was a known intermediate in glycolysis, but what was the fate of the remaining 2-carbon fragment? Polly Smyrniotis and I, now joined by my first foreign postdoctoral fellow, Hans Klenow from Copenhagen, set out to purify the enzyme(s) involved in the cleavage of pentose phosphate, using rat liver as the enzyme source and an assay that measured the appearance of glyceraldehyde 3-phosphate. When we also followed the disappearance of the 5-carbon sugar, using Dische's “orcinol” reaction, we detected the formation of a new product that also reacted with orcinol but produced a different color and a visible absorption spectrum distinctly different from that produced in the reaction with pentoses. Our clue to the identity of this product came from Melvin Calvin's laboratory in Berkeley, where, using radioactive carbon dioxide, they had identified both ribulose diphosphate and a 7-carbon sugar, sedoheptulose monophosphate, as early intermediates in the fixation of carbon dioxide in photosynthesis. Authentic sedoheptulose was available from Nelson Richtmyer's laboratory in Building 4, and on Christmas Eve, 1951, when everybody else had gone home, I sprayed a paper chromatogram with orcinol and up came the blue spot characteristic of sedoheptulose. I rushed up and down the laboratory hallway clutching the paper chromatogram, but there was nobody there to show it to, so I took it home and hung it on the Christmas tree, singing the little ditty: “It's sedoheptulose, it's sedoheptulose, tra la la boom deay, tra la la boom deay,” much to the amusement of my young daughters. We adopted the name “transketolase,” first suggested by Racker and his co-workers, because it catalyzed the transfer of a 2-carbon fragment from the ketopentose, ribulose 5-phosphate, to the other 5-carbon sugar, ribose 5-phosphate, to generate the new ketol linkage in the 7-carbon sugar, sedoheptulose 7-phosphate. Thus the unknown 2-carbon fragment never occurred as a free entity. Later, simultaneously with Racker, we showed that the coenzyme carrier for the 2-carbon fragment by transketolase was thiamine pyrophosphate. Two puzzling observations remained to be explained. One was that the configuration of the hydroxyl group on the third carbon atom of the new product, sedoheptulose 7-phosphate, was opposite that on the third carbon atom of the presumed substrate, ribulose 5-phosphate. This lack of stereospecificity, particularly because we had demonstrated that the reaction was readily reversible, was highly improbable for an enzyme-catalyzed reaction. The other unexplained observation was that the cleavage of pentose phosphate by our purified transketolase preparations from rat liver required the presence of aldolase, a crystalline and supposedly pure enzyme from rabbit muscle that catalyzed the condensation of two triose phosphates to form the hexose, fructose 1,6-bisphosphate. The answer to both of these puzzling observations came from the discovery by Paul Stumpf, while on sabbatical in my laboratory, and reported almost simultaneously from the laboratories of Dickens, Racker, and Ashwell, of another enzyme, an “epimerase,” that catalyzed the conversion of ribulose 5-phosphate to its “3-epimer,” xylulose 5-phosphate, which had the same stereo-configuratuion at the 3-carbon atom as sedoheptulose phosphate. With this substrate, we confirmed the earlier report by Racker and his co-workers that xylulose phosphate, rather that ribulose phosphate, was the true substrate for cleavage by transketolase and the true donor of the 2-carbon fragment. The requirement for aldolase was explained when we found that crystalline preparations of this enzyme from rabbit muscle contained the epimerase as a contaminant, which could only be removed by many re-crystallizations. Thus three different pentose phosphates were now shown to be involved in the new pathway: ribulose 5-phosphate, the first product of the oxidation of 6-phosphate gluconate, and xylulose 5-phosphate and ribose 5-phosphate, both formed from ribulose 5-phosphate, one serving as the 2-carbon donor and the other as the acceptor in the reaction catalyzed by transketolase. The addition of any one of these pentose phosphates to crude tissue extracts would result in the formation of an equilibrium mixture of all three. Still to be discovered, however, was a mechanism that would the products of the transketolase reaction, sedoheptulose phosphate and glyceraldehyde phosphate, to the 6-carbon fructose 6-phosphate and glucose and complete the cycle. In what was the fate of sedoheptulose We found that purified enzyme preparations from liver or yeast would catalyze the formation of hexose from sedoheptulose but only if triose phosphate was also When I this finding at one of our journal who was from the University of at and working in Kornberg's laboratory, suggested that we consider the of another this of a 3-carbon fragment from sedoheptulose phosphate to triose phosphate to generate fructose When we out an with triose phosphate, we found as the fructose 6-phosphate formed had in the three carbon atoms with the first three We the enzyme because it catalyzed the transfer of an linkage rather the cleavage catalyzed by What remained was to for the fate of the remaining 4 carbon atoms of sedoheptulose 7-phosphate. this work Polly Smyrniotis and I were joined by Paul and two young working as in the new in Building who had asked to join my group to enzymology in their which out to be from to We identified the missing fragment as another new sugar ester, the sugar in a number of including its conversion to the 7-carbon sugar sedoheptulose in a condensation with phosphate, catalyzed by fructose It was also converted to fructose 6-phosphate in the reaction catalyzed by transketolase. The of the pentose phosphate pathway had now been It of two an oxidative in which the hexose, glucose was converted to pentose phosphate and carbon dioxide with the reduction of two of TPN, and a in which three of pentose phosphate carbon were to two and of hexose phosphate carbon in a of Our contributions included the discovery of three new sugar phosphate ribulose 5-phosphate, sedoheptulose 7-phosphate, and and three new and pentose-phosphate We with Racker the discovery of transketolase and confirmed his finding that xylulose 5-phosphate, rather ribulose 5-phosphate, was the 2-carbon donor in the reaction catalyzed by that We also with and Dickens, working in England, the discovery that fructose 6-phosphate was also a substrate for transketolase. the pathway as turns of the would result in the oxidation of one molecule of 6-carbon sugar to of carbon The of the new however, out to be different from the pathway for the direct oxidation of carbohydrate that we had It two for the of ribose 5-phosphate. One is the of the which also 2 of 5-phosphate also be formed from hexose and triose phosphates by the catalyzed by transketolase and of as in the of or the pentose phosphates produced would be back to hexose in the the following carbohydrate pentose to make nucleotide, that is not the to the pentose phosphate With Six of to and the back the a pathway the pentose phosphate is reduced been The is then to make the to its metabolic the oxidative now by An intermediate by of Six three set ribulose 5-phosphate formed from three of One from to ribulose 5-phosphate to the phosphate of ribose The other two at Two xylulose formed called Two from from the to as everybody Thus three made to from sedoheptulose the for number two two to their transfer as a made another a triose phosphate what we now by two by glucose when we out with The first discovery to the new pentose phosphate the formation of ribulose and ribose phosphates as products of the oxidation of 6-phosphogluconate, was in the of 1952 at the meeting of the American Chemical Society in Chicago. The of the complete pentose phosphate including the reactions catalyzed by the new enzymes transketolase and was published in in a in with and for entitled of in The of the cycle in mammalian liver and in was confirmed in experiments with ribose 5-phosphate in two papers published with of the National Laboratory, work out there during the of 1953. in 1952, Calvin's group at the University of at reported evidence for ribulose as the acceptor for the formation of acid, the first fixation product in we were by the that the pentose phosphate pathway serve as the mechanism for this key intermediate from hexose a arrived postdoctoral student from and Polly Smyrniotis out the first experiments to identify the involved. They were able to show that with crude extracts from ribose 5-phosphate was a substrate for the formation of acid, and they purified a from that they used to the of ribulose In the fall of we from Building 3 to new laboratories on the of the NIH where, joined by we isolated the enzyme responsible for that reaction, as well as the enzyme which catalyzed the formation of 2 of acid from ribulose and in laboratories the from each and I was with the purification of I took on the of pure ribulose and after the most important enzyme, the The a the enzyme was purified only from the crude by all the that we could it appeared to be a pure which that it about of the in the work in other laboratories the confirmed this and this enzyme, now known as for is now to be the most on Our work was published in three papers in the of the Journal of Biological Chemistry, “The and of and “The of from and With this work and our earlier demonstration of the reactions for the interconversion of pentose and hexose with sedoheptulose phosphate as a all of the enzymes for the reactions of the were The pentose phosphate pathway in as two important cell for ribose 5-phosphate for the of nucleotides and and for in the form of In it to the ribulose from the hexose phosphates to required for the of ribulose from ribulose 5-phosphate and also for the reduction of acid to glyceraldehyde 3-phosphate. The for the reaction, is also by the action of in the In both and rather to function as the coenzyme for