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Introduction...509 Notes on some of the most exceptional investigators...513 Fermentation by yeast extracts...517 The rôle of phosphates in fermentation...518 The discovery of NAD and NADP...524 The formation of glycerol in fermentation...525 Recognition of an identical glycolyticpathway in yeasts, animals and plants...529 Elucidating some enzymes of alcoholic fermentation...531 Conclusion...536 References...537 Scientists do not solve problems because they possess a magic wand … but because they have studied a problem for a long time … (Feyerabend, 1975 [67] p. 302). Probably most species of yeasts can ferment sugar to ethanol 239. They are famous for this ability, especially on an industrial scale, and this is why research on fermentation by yeasts has had extensive financial support. The second and third articles in this series1 describe how, in the nineteenth century, Louis Pasteur2 carried out extensive physiological studies of fermentation by intact living yeast cells and later, in 1897, Eduard Buchner 31, 32 achieved fermentation by cell-free extracts, making it practicable to study the biochemistry of fermentation in vitro 19, 20. The present review records how the metabolic pathway of alcoholic fermentation was gradually revealed (summarized chronologically in Table 1). During the twentieth century, this research was central for generating major advances in biochemistry, with massive economic applications. … the initiation of fermentation does not require so complicated an apparatus as the living cell. The agent responsible for the fermenting activity of the extracted juice is a dissolved substance, no doubt a protein; this will be called zymase.3 This is the catabolic pathway5 by means of which D-glucose is broken down to pyruvate to produce two moles of ATP6 per mole of glucose (Figures 1 and 2). In alcoholic fermentation, yeasts convert the pyruvate to ethanol and carbon dioxide and this whole process gives the yeasts chemical energy which is stored in the phosphate bonds of ATP 139. ATP was discovered in 1929 in animal tissues by Karl Lohmann 142 and also simultaneously by Cyrus Fiske7 and Yellapragada Subbarow8 71. However, its rôle as a phosphate donor in the formation of hexose phosphates and its importance in many other enzymic reactions was not then recognized. The glycolytic pathway. Each reaction of the pathway is given a letter for reference in the text. Note: Because one molecule of D-fructose 1,6-bisphosphate yields two molecules of glyceraldehyde 3-phosphate (D, E), thereafter there are two molecules of each catabolite for each molecule of D-glucose phosphorylated Path of carbon atoms in the conversion of glucose to ethanol and carbon dioxide. Each carbon atom of a glucose molecule is numbered to show its fate during fermentation It is interesting to speculate on how enzymology might have developed if the simple experiment to prepare a cell-free yeast extract and to prove the enzymic nature of fermentation (for which the relatively modest equipment needed was then available) had been carried out as an immediate sequel to the work of Cagniard-Latour, Schwann and Kützing. The eventual upsurge of enzymology could have occurred at least 50 years earlier … (224 p. 254). Enzymes are neither proteins, nor carbohydrates, nor do they belong to any of the known large groups of complex organic compounds.9 If, as many workers believe, the enzymes are all proteins, it is certainly remarkable that the majority of the successful attempts to purify them have led to the obtaining of substances which are at least predominantly non-proteins, although the original material from which they were derived consisted largely of protein (90 pp. 174–175). The only regrettable point in Pasteur's work on fermentation is that he did not explore Traube's suggestion of enzyme action in the yeast cells, nor did he visualize the possibility of extracting fermentation enzymes, even though an ever-increasing number of cell-free enzyme actions were being reported. Pasteur's chemical training and experimental skill would have given a high chance of success to such experiments (224 p. 253). The catabolism of pyruvate to ethanol by yeasts, or to lactic acid by muscle By 1940, the complete pathway of glycolysis had been elicited, largely by a few remarkable biochemists, of whom five were of Jewish origin, as was an astonishing number of other outstanding twentieth century biochemists. Six were Nobel prizewinners. In the 1930s and 1940s, a number of such notable scientists and their colleagues became victims of political turbulence and social upheaval, and so were forced into exile. Several were refugees from the German Nazi government of the 1930s 53 and contributed enormously to the advances of biochemistry in the countries where they settled, particularly Britain and America (which were at war with Germany in the 1940s until 1945). The following are brief notes on the lives of some outstanding biochemists who elucidated the glycolytic pathway. Carl Ferdinand Cori (1896–1984) was born in Prague (then within the Austro-Hungarian Empire), spent much of his youth in Trieste, and studied medicine in Budapest and Prague. He married Gerty Radnitz (see below). When working in the University of Graz in 1922, he decided to emigrate to the USA, partly because of the poverty in Austria at that time (an effect of the Treaty of Versailles) and partly, as his wife was Jewish, because of local anti-semitism (it was required to prove 'Aryan' descent to be employed at the university). On invitation, he went to work in Buffalo, New York, moving to Washington University medical school in 1931. Carl and Gerty Cori jointly received the Nobel Prize for Physiology or Medicine in 1947. Like many others, Carl was a dedicated experimenter and felt strongly about administrative work. He wrote '… Faustus considers suicide … [but survives] by making a pact with the devil, who promises him power … a similar crisis exists when a scientist begins to play with the idea of going into administration' (46 p. 1) 41, 192, 209. Gerty Theresa Cori (née Radnitz) (1896–1957), like her husband Carl, was born in Prague, where she too studied medicine. She emigrated to the USA with Carl, with whom she worked closely thereafter. Gerty Cori was only the third woman to receive a Nobel prize in science, the others being Marie Curie and Irène Joliot-Curie 75, 193. Gustav Embden (1874–1933), studied medicine at the universities of Freiburg-im-Breisgau, Munich and Strasbourg, later working with Paul Ehrlich at Frankfurt. Embden became professor and, in 1925, rector of Bonn University. Working with muscle, he made his very significant contributions to research on glycolysis 47, 226. Harden's outstanding qualities as an investigator were clarity of mind, precision of observation, and a capacity to analyse dispassionately the results of an experiment and define their significance. He mistrusted the use of his imagination beyond a few paces in advance of the facts. Had he exercised less restraint, he might have gone further; as it was he had little to withdraw 113. Arthur Harden. © The Nobel Foundation, reproduced by permission Otto Fritz Meyerhof (1884–1951) (Figure 5) qualified in medicine at Heidelberg, having written a thesis on a psychiatric subject, and was actively interested in philosophy for much of his life. In 1918 Meyerhof chose muscle for experimental work, because it then seemed the most convenient and promising material to study the connexions between chemical changes, heat production and mechanical work 176. He was at the Kaiser Wilhelm Institute for Experimental Therapy and Biochemistry, Berlin14 from 1924–1929, when he became head of the department of physiology at the Kaiser Wilhelm Institute for Medical Research in Heidelberg. With the Nazis in power Meyerhof, being Jewish, had to leave Germany and so worked in Paris from 1938 to 1940. Then, when the Germans occupied Paris, he fled to the USA, becoming professor at the University of Pennsylvania. He was welcomed there, having shared the 1922 Nobel Prize in physiology or medicine with A. V. Hill 77, 202. Otto Fritz Meyerhof. © The Nobel Foundation, reproduced by permission Carl Neuberg (1877–1956) (Figure 6), although one of the main founders of modern biochemistry, had a less illustrious scientific career than that of Meyerhof. In 1906, he started the Biochemische Zeitschrift and edited 278 volumes over the next 30 years. He became director of the Kaiser Wilhelm Institute for Experimental Therapy and Biochemistry, Berlin, in 1925 and it is said that his laboratory generated about 900 publications (78 p. 272); but, as he was Jewish, the Nazi regime forced him to leave the Institute and he emigrated to The Netherlands, to Palestine and, finally to the USA in 1940. Like many others, his career reflected the political upheavals of his time 65, 84, 140, 189, 191. Carl Neuberg. Photograph reproduced by kind permission of Archiv zur Geschichte der Max-Planck-Gesellschaft, Berlin–Dahlem Jacob Karol Parnas (sometimes called Yakub Oskarovich Parnas) (1884–1949) also had a life much affected by the political geography of the twentieth century. He was born in a part of the Austro-Hungarian Empire, near the border of what was then Russian Poland, but is now the Ukraine. He, too, was of Jewish descent, his native town, Tarnopol, having about 30 000 inhabitants, half of whom were Jews 3. Parnas held professorships in Strasbourg (1913), then a part of Germany, now in France; in Warsaw (1916–1919), which was then in Russia, but now Poland; and in Lwów (1920–1941), then in Poland, but now Lviv in the Ukraine. From 1943, he was head of the Biological and Medical Chemistry Institute in Moscow 125 (227 pp. 434–435). Hans Karl August Simon von Euler-Chelpin (1873–1964), who published as H. von Euler, was, like Harden, a polymath of great versatility. He studied painting at the Munich Academy and then physics in Berlin under Max Planck and organic chemistry under Emil Fischer. Later, von Euler worked in Göttingen with Walther Nernst, also in Stockholm with Svante Arrhenius and, back in Berlin, with Jacobus van't Hoff. Although born a German, he became a Swedish citizen in 1902 and was professor of chemistry at Stockholm from 1906; yet von Euler served in the German armed forces in World War I and later, evidently unmoved by Hitlerism, as a German diplomat during World War II. In 1929, he shared the Nobel prize in chemistry with Harden for work on fermentation. His son, Ulf von Euler, also became a Nobel prize-winner, in medicine or physiology 115, 188. Otto Heinrich Warburg (1883–1970) (Figure 7), one of the greatest of all biochemists, took a doctorate under Emil Fischer in Berlin. He was in the Prussian army in World War I but spent most of his working life at the Kaiser Wilhelm Institute for Cell Physiology, Berlin. As well as an enormous output of over 500 publications, mostly on cell metabolism, on which subject he made major contributions, Warburg was responsible for significant advances in biochemical methodology. The Warburg manometer, developed for measuring rates of gas exchange in the 1920s, became standard equipment in biochemical laboratories from the 1930s to the 1960s. The gas phase in the manometer vessel (Figure 8) was achieved by constant shaking of the vessels in a temperature-controlled water bath 246. Warburg was also responsible for valuable developments in spectrophotometry and received the Nobel prize for physiology or medicine in 1931. Otto Heinrich Warburg. Photograph reproduced by kind permission of Archiv zur Geschichte der Max-Planck-Gesellschaft, Berlin–Dahlem The Warburg manometer. The U-tube (T) of narrow bore is calibrated in millimetres. The bottom of the tube is attached to a rubber reservoir (R) and the screw clamp squeezes the reservoir and thereby adjusts the level of the liquid in the tube. The left arm of the tube is open at the top; the right arm has a side arm (S) to which a glass vessel can be attached by means of a ground joint. At the top of the right arm is a tap, by which the vessel can be closed or opened. The manometer is mounted on a board which can be attached to a shaking apparatus. Reproduced by permission from Krebs (1981 127) … I learned that a scientist must have the courage to attack the great unsolved problems of his time, and solutions usually have to be forced by carrying out innumerable experiments without much critical hesitation (248 p. 1). Despite his Jewish ancestry, Warburg was not persecuted by the Nazis, as he was protected by Reichsmarschall Göring (Goering), who ruled that Warburg was to be unharmed as he was only one-quarter Jewish.15 Much of Warburg's research was on cancer, which was a source of great anxiety to the leading Nazis 126, 127. After Buchner's success with fermentation by cell-free yeast extracts in the first years of the twentieth century, it was deemed necessary to find out how, if at all, such fermentation differed from that by intact living cells. Using brewing yeasts, three kinds of cell-free preparation that ferment sugars were used quite widely: (i) Buchner's 'zymase', described in the third of the present articles 20, was made by grinding the yeast mixed with quartz sand and kieselguhr; (ii) in 1900 Robert Albert16 prepared 'Zymin' by repeatedly treating yeast with acetone 1, 2; (iii) a product was obtained by macerating dried yeast 131; this preparation was called Lebedew17 juice, for example by Maurice Ingram18 117 and by Friedrich Nord19 and Sidney Weiss20 190, while Joseph Fruton21 refers to 'juice of Lebedev' (80 p. 295). The term 'zymase' was sometimes used for Buchner's whole yeast extract 38 and sometimes for the 'enzyme' present in yeast extract and responsible for converting sugar to ethanol and carbon dioxide. Haldane used the word 'myozymase' for 'the glycolytic enzyme complex of muscle' (90 p. 133). A fourth technique for obtaining active cell-free extracts of yeasts, although perhaps not much used, was developed in 1913 by Henry Dixon22 and William Atkins23, who extracted 'zymase' from brewery yeast by freezing the yeast in liquid air 54. In 1911, Harden reported that living yeast (intact cells) ferments glucose 'forty times as quickly' as yeast juice (94 p. 27). He had improved on the method of Allan Macfadyen24 and his colleagues at the Jenner Institute in London, who had already estimated the carbon dioxide evolved in yeast fermentation by passing the gas through sodium hydroxide and titrating 149. Harden was subsequently able to make more frequent measurements of fermentation with an azotometer (or 'nitrometer') 221 (Figure 9); this equipment enabled him to take readings of carbon dioxide production about every 4 min. Harden's use of a Schiff's azotometer 221 connected to a fermentation flask. The medium is first saturated with carbon dioxide; the volume of gas evolved can then be measured. The level of mercury in the reservoir is kept constant by a syphon overflow so that no change of pressure in the flask occurs. For each reading, the fermenting mixture must be shaken vigorously in order to avoid supersaturation with carbon dioxide. Reproduced by permission from Harden, Thompson and Young (1910 [100]) … the confusion in the literature as to the quantitative relations of lactic acid in muscle was wholly due to faulty technique …. When the muscle is disintegrated as a preliminary to extraction for analytical purposes, the existing equilibrium is entirely upset (112 p. 361). Many workers studied the rôle of phosphates in glycolysis during the first half of the twentieth century. Their research not only uncovered the course and nature of alcoholic fermentation by yeasts and of lactic acid production by muscles; it was also the key to understanding other metabolic processes, including the energy-transforming machinery of living cells. In 1870, the extremely distinguished German chemist, Adolf von Baeyer,27 offered a wild speculation 15 that the intermediate stages of ethanolic and lactic acid fermentations involved the successive removal and addition of water (Figure 10). Thirty-one years later, an of for the glycolytic pathway was made by who from (then part of In that phosphates fermentation p. and Buchner this p. published in 1870, of intermediate stages of ethanolic fermentation from glucose the successive removal and addition of water to its and from removal of water and of in the of the carbon The in lactic acid fermentation to acid and in alcoholic fermentation to der der … 15 p. The that yeast is able to effect the fermentation of a relatively part of the sugar has usually been to the action of a enzyme of the It was of to study the effect of to the mixture of yeast and … was the of a of attempts to a similar effect by in the course of which a and of was used … (94 p. The two to which the in fermentation by the addition of juice were were the of phosphates in the and the in yeast juice of a or the of which is for fermentation (94 p. … the of carbon dioxide and which would have been in the of phosphate by a to the phosphate in the or (94 p. … solutions of sodium or phosphate … or a mixture of with the were … the liquid being to the was saturated with carbon dioxide at the of the and the volume of carbon dioxide by the addition of acid was … The of carbon dioxide evolved each addition is the and is … to the phosphate (see and pp. by Harden and Young in the action of phosphate on the fermentation of glucose by yeast The mixture water glucose at A no phosphate and successive of of sodium phosphate or From of … It that the of phosphate is for the alcoholic fermentation of glucose by the reaction which being the The is then of phosphate exists which a of fermentation. of beyond this the of fermentation p. 1,6-bisphosphate In an to their Harden and Young from the by acid in and by enzyme action in its chemical was not until by and at the Institute As as Harden made a which subsequently is remarkable that the is not or by living p. there are now known to be no in the of (i) yeasts sugar and (ii) glycolytic do not from the metabolic in Harden and Robert D-glucose from fermenting yeast juice years later, Neuberg discovered hexose D-fructose by D-fructose 1,6-bisphosphate and this was subsequently in fermenting yeast juice developed by the in the 1920s, made it to the of phosphates in the By and obtained D-glucose as is the of the intermediate of some one of the hexose it to at the to it into with some of the … The production of of the with an of formation of and it that this be but a part of the intermediate product which has the Meyerhof and … by his for many years an to a of the of p. of bonds in glycolysis Krebs and in Warburg and and studied the activity of at Warburg had developed the use of cells and for measuring enzyme activity and discovered that have a studies enabled Warburg and his colleagues to the nature of the of ATP in the With the they could show that glyceraldehyde 3-phosphate was certainly the in the Fritz first to the bonds of ATP in 139. This is not the needed to a between two but of bonds much This energy is used in in active and in Harden and Young made that glucose fermentation on the of a material in their yeast extracts pp. Their was a first understanding the rôle of for enzymic This was a major in … the experiment was made of carrying out the fermentation in the of with the that about to per more sugar was than in the of the … This … was the of … attempts to a similar effect … in the course of which a and of was used … to produce a very in the (94 p. A between research on glycolysis in yeast and in muscle was reported in two of 1918 He had in and other animal the which had been in alcoholic fermentation. In he the to be necessary for as well as metabolism, by yeast and A number of years the of Harden and were into and In the was to have two one of which Meyerhof and Lohmann as ATP The other was by von Euler and his colleagues and by Warburg and and was to be a of and with two phosphate groups Warburg and that its be and for the of a which they had from yeast extract in (see Table Warburg and the active part the as in Because Warburg that von Euler and were on the with their from he did not like idea of going to Stockholm for He finally but going to if the word in p. The of the industrial production of glycerol from yeast fermentation is a remarkable example of how studies in this and industrial can This example of was to great use by Germany in World War when the for glycerol for making the the which from the a century had of glycerol to be when yeast ferments sugar to ethanol and carbon dioxide. In 1913 and workers Buchner and of sugar by yeast juice and and reported the formation of acid during alcoholic fermentation. When Otto and acid to be by yeast to ethanol and carbon and that pyruvate might be an intermediate in ethanolic fermentation, colleagues to leave the study of the rôle of acid in sugar fermentation for his to have been many others, went on to on the part by pyruvate in fermentation. between and Neuberg and his colleagues finally (i) pyruvate is during hexose (ii) the pyruvate is to and carbon dioxide; and (iii) is to by to fermenting and so an addition with they that hexose is broken down to which are of is the of which down to acid and the of the is the only that can be made is the formation of of the fermentation pathway by which glycerol is of the fermentation pathway by The German chemical in Berlin, where and the process in Germany in and the process to By this in Germany during World War at least of glycerol were every and used to make for The was about of the sugar p. … during the the German army was interested in the experiments and results work from the of the time and the that the of glycerol to the would be because of the … for some time have been with the between catabolism in yeast and catabolism of in for the fermentation published in pp. Meyerhof that lactic acid was from or by muscle extracts if yeast were He used the word for this which he obtained by ethanolic of Then, in Warburg and from yeast an enzyme from which von Euler and later obtained two 1, with ATP and the of hexose or to and Much lactic acid was the mixture had been for and and to muscle The study of phosphate in muscle extracts a into the of the of fermentation, because the to this formation of are entirely identical in the of alcoholic fermentation and lactic acid production … between the glycolytic of muscle and the of yeast are revealed by the similar action of chemical substances on the two For Harden that strongly fermentation in extracts of yeast on of of the … From and other must that with phosphate is the of the alcoholic fermentation and the formation of lactic acid and, that the is most the next in the … be the in there is a