A history of research on yeasts 5: the fermentation pathway
2003; Wiley; Volume: 20; Issue: 6 Linguagem: Inglês
10.1002/yea.986
ISSN1097-0061
Autores Tópico(s)Yeasts and Rust Fungi Studies
ResumoIntroduction...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 observation of particular significance for elucidating the glycolytic pathway was made by Augustyn Wróblewski,28 who came from Vilnius, Lithuania (then part of Russia). In 1901, Wróblewski found that certain phosphates accelerate fermentation (264 p. 12) and Buchner soon confirmed this finding (34 p. 142). Baeyer's scheme, published in 1870, of intermediate stages of ethanolic fermentation from glucose involving the successive removal and addition of water. I, Hydrated aldehydo-glucose (i.e. water added to its aldehyde carbonyl group). II and III, possibly intermediates resulting from removal of water and re-addition ('accumulation of oxygen resulting in the fission of the carbon chain'a). The fission occurs once in lactic acid fermentation to give IV (lactic acid anhydride) and thrice in alcoholic fermentation to yield V, diethyl dicarbonate [aDie Folge der Accumulation des Sauerstoffs ist die Sprengung der Kohlenstoffkette … 15 p. 74.] The fact that yeast press-juice is able to effect the fermentation of a relatively small part of the available sugar has usually been attributed to the action of a proteolytic enzyme of the juice. It was therefore of considerable interest to study the effect of adding serum to the mixture of yeast press-juice and sugar29. … was the starting-point of a series of attempts to obtain a similar effect by different means, in the course of which a boiled and filtered solution of autolysed yeast-juice was used … (94 p. 38). The two factors to which the increase in fermentation produced by the addition of boiled juice were ultimately traced were (1) the presence of phosphates in the liquid, and (2) the existence in boiled fresh yeast juice of a co-ferment or co-enzyme [NAD], the presence of which is indispensable for fermentation (94 p. 39). … the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO2 or (94 p. 40). … solutions of sodium or potassium phosphate … monohydrogen salts or a mixture of these with the dihydrogen salts were always used. … the liquid before being added to the yeast-juice was saturated with carbon dioxide at the temperature of the bath, and the volume of carbon dioxide liberated by the addition of excess hydrochloric acid was ascertained … The extra amount of carbon dioxide evolved after each addition is the same, and is equivalent … to the phosphate added (see Figure 11 and 103 pp. 414, 415–416). Illustration by Harden and Young in 1905 showing the action of phosphate on the fermentation of glucose by yeast extract. The mixture contained 25 cm3 yeast-juice + 20 cm3 water + 10% glucose at 25 °C; A no phosphate added; B and C successive additions of 5 cm3 of 0.3 M sodium phosphate (Na2HPO4 or Na2HPO4 + NaH2PO4). From Figure 3 of 103 … It appears probable that the presence of phosphate is essential for the alcoholic fermentation of glucose by yeast-juice, the reaction which occurs being the following: The hexosephosphate thus formed is then hydrolysed: An optimum concentration of phosphate exists which produces a maximum initial rate of fermentation. Increase of concentration beyond this optimum diminishes the rate of fermentation (105 p. 311). D-Fructose 1,6-bisphosphate (the Harden–Young ester) In an attempt to characterize their ester, Harden and Young isolated fructose from the ester, both by acid hydrolysis in 1909 265 and by enzyme action in 1910 106. But its chemical structure (D-fructose 1,6-bisphosphate) was not determined until 1928, by Phoebus Levene33 and Albert L. Raymond34 at the Rockefeller Institute 138. As early as 1914, Harden made a comment which subsequently proved highly pertinent: 'It is remarkable that the hexosephosphate is not fermented or hydrolysed by living yeast…' (95 p. 51). Indeed, there are now known to be no hexose-phosphate carriers in the plasma membranes of yeasts. Hence (i) yeasts cannot utilize exogenously supplied sugar phosphates, and (ii) glycolytic intermediates do not leak from the cells—a metabolic economy. Also in 1914, Harden and Robert Robison35 isolated D-glucose 6-phosphate ('a hexosemonophosphoric acid') from fermenting yeast juice 99. Four years later, Neuberg discovered another hexose monophosphate, D-fructose 6-phosphate (the 'Neuberg ester'), by hydrolysing D-fructose 1,6-bisphosphate 182; and this ester was subsequently detected in fermenting yeast juice 214. Methods developed by the carbohydrate chemist Walter Haworth36 in the 1920s, made it possible to determine the structures of these various phosphates in the early 1930s. By these means, Robison and Earl King37 obtained pure D-glucose 6-phosphate ('an aldosemonophosphoric ester') 215. Attractive as is the theory of the intermediate character of some one of the hexose phosphates, it seems to me impossible at the moment to bring it into agreement with some of the facts … The production of 70–80% of the monophosphate, with an unaltered degree of formation of alcohol and CO2, renders it impossible that this ester should be 'obviously nothing but a part of the intermediate product which has escaped the coupled decomposition–esterification reaction'38 (O. Meyerhof and K. Lohmann, Biochem. Z., 185 (1927) 155). … Hardens's views, popularized by his widely-read book 94, remained for many years an obstacle to a correct interpretation of the meaning of phosphorylation (74 p. 55). Formation of energy-rich bonds in glycolysis (after Krebs and Kornberg 129) Then in 1939 Warburg and Walter Christian42 253 isolated, crystallized and studied the activity of glyceraldehyde-3-phosphate dehydrogenase, using spectrophotometric absorption at 340 nm. Warburg had previously developed the use of photoelectric cells and monochromatic light43 for measuring respiratory enzyme activity 257, 258 and discovered that reduced pyridine nucleotides have a λmax ≈ 340 nm 251. These studies enabled Warburg and his colleagues to unravel the nature of the involvement of ATP in the above reaction. With the pure crystalline enzyme, they could show that glyceraldehyde 3-phosphate was certainly the substrate in the coupled oxido-reduction–phosphorylation. Fritz Lipmann44 first referred to the 'energy-rich' pyrophosphate bonds of ATP in 1941 139. This is not the 'bond energy' needed to break a bond between two atoms; but hydrolysis of these energy-rich bonds releases much free energy. This energy is used in biosyntheses, in active transport across membranes and in muscular contraction. Harden and Young made another seminal finding: that glucose fermentation depended on the presence of a heat-stable, dialysable material in their yeast extracts (103 pp. 410–413). Their observation was a first step towards understanding the crucial rôle of co-enzymes for certain enzymic activities. This was a major development in biochemistry. … the experiment was made of carrying out the fermentation in the presence of serum, with the result that about 60 to 80 per cent. more sugar was fermented than in the absence of the serum … This … was the starting-point of … attempts to obtain a similar effect … in the course of which a boiled and filtered solution of autolysed yeast-juice was used … [and] found to produce a very marked increase in the total fermentation… (94 p. 38). A further close association between research on glycolysis in yeast and in muscle was reported in Meyerhof's two papers of 1918 151, 152. He had detected, in muscles and other animal tissues, the co-enzyme which had been found in alcoholic fermentation. In addition, he showed the co-enzyme to be necessary for respiration, as well as anaerobic metabolism, by both yeast and muscle. A number of years passed before the components of Harden and Young's 'coferment' were resolved into NAD, ATP, thiamine pyrophosphate48 ('cocarboxylase') and Mg2+. In 1931, the heat-stable fraction was shown to have two constituents, one of which Meyerhof and Lohmann identified as ATP 143, 168. The other was highly purified by von Euler and his colleagues and separately by Warburg and Christian and was found to be a dinucleotide of adenine and nicotinamide with two phosphate groups 225, 251. Accordingly, Warburg and Christian 250 suggested that its name should be Diphospho-Pyridinnucleotid and proposed Triphospho-Pyridinnucleotid for the co-factor of a hexose-monophosphate dehydrogenase which they had isolated from yeast extract in 1932 (see Table 3). Warburg and Christian crystallized the active part [of the coferment, NADP] as picrolonate in December, 1933. Because Warburg suspected that von Euler and Myrbäck were on the same track with their cozymase from yeast, he did not like my idea of going home to Stockholm for Christmas. He finally agreed, but advised me, 'I am going to kill you if you mention the word 'picrolonic acid' in Stockholm' (236 p. 152). The story of the industrial production of glycerol from yeast fermentation is a remarkable example of how academic studies (of glycolysis, in this case) and industrial practice can interact advantageously. This early example of biotechnology was put to great practical use by Germany in World War I, when the demand for glycerol (needed for making the powerful explosive nitroglycerine50) exceeded the supply which normally came from the soap industry. Half a century before, Pasteur 201 had shown small amounts of glycerol to be produced when yeast ferments sugar to ethanol and carbon dioxide. In 1913 and 1914, several workers confirmed these observations. Buchner and Meisenheimer51 36 found 3.8% of sugar fermented by yeast juice forms glycerol; and Fernbach and Schoen 66 reported the formation of pyruvic acid during alcoholic fermentation. When Otto Neubauer52 and Konrad Fromherz53 found pyruvic acid to be converted by yeast to ethanol and carbon dioxide, and suggested that pyruvate might be an intermediate in ethanolic fermentation, Neubauer wrote: 'I ask colleagues to leave the continued study of the rôle of pyruvic acid in sugar fermentation for us …'54 Alas, his plaintive plea seems to have been ignored. Neuberg, among many others, went on to publish numerous papers on the part played by pyruvate in fermentation. Indeed, between 1910 and 1920 Neuberg and his colleagues finally established that: (i) pyruvate is formed during hexose fermentation; (ii) the pyruvate is decomposed to acetaldehyde and carbon dioxide; and (iii) acetaldehyde is reduced to form ethanol. Furthermore, by adding sulphite to fermenting yeast, and so forming an addition compound with acetaldehyde, they confirmed that hexose is broken down to C3 compounds, which are derivatives of glycerol. Glycerol is the reduction equivalent of pyruvic acid, which breaks down to carbonic acid and acetaldehyde. If the reduction of the latter is blocked, the only adjustment that can be made is the increased associated formation of glycerol.60 Neuberg's modification of the fermentation pathway by which glycerol is produced. (A) Summary of the fermentation pathway; (B) pathway modified by adding sulphite The German chemical factory, Vereinigte Chemische Werke AG in Berlin, where Connstein and Lüdecke worked, patented the sulphite process in Germany in 1915 4, 44 and adapted the process to manufacture glycerol. By this means, in Germany during World War I, at least 106 kg of glycerol were manufactured every month and used to make nitroglycerine for explosives. The yield was about 15–20% of the sugar fermented (98 p. 131). … because, during the war, the German army administration was interested in keeping the experiments and results secret. Our work stemmed from the needs of the time and the expectation that the supply of glycerol available to the European Central Powers would soon be inadequate, because of the blockade.61 … for some time we have been busy with developing the analogy between fermentative catabolism in yeast and anaerobic catabolism of carbohydrates in muscle.63 Oppenheimer's scheme for the fermentation pathway, published in 1926 (194 pp. 428–462) Meyerhof 156 found that lactic acid was produced from glucose, fructose or mannose by muscle extracts if yeast 'activator' were added. He used the word 'hexokinase' for this 'activator', which he obtained by ethanolic precipitation of autolysed yeast. Then, in 1932, Warburg and Christian isolated from yeast an enzyme fraction, Zwischenferment 250, from which von Euler and Erich Adler65 242 later obtained two fractions. Fraction 1, with added ATP and Mg2+, catalysed the phosphorylation of hexose (glucose or fructose) to give hexosemonophosphate and adenylic acid. Much lactic acid was produced after the mixture had been incubated for 15–30 min and boiled, cooled and added to muscle extract. The study of phosphate esterification in muscle extracts provides a deeper insight into the mechanism of the Harden–Young equations of fermentation, because the phenomena related to this formation of ester are entirely identical in the cases of alcoholic fermentation and lactic acid production … Further similarities between the glycolytic function of muscle and the fermentative function of yeast are revealed by the similar action of certain chemical substances on the two processes. For example, Harden showed that arsenates strongly accelerate fermentation in extracts of yeast on account of quicker splitting of the hexose-diphosphoric ester … From these and other analogies we must conclude that esterification with phosphate is the common initial stage of both the alcoholic fermentation and the formation of lactic acid and, moreover, that the same three-carbon compound is most probably the next stage in the decomposition … Whatever may be the differences in detail, there is a surprising
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