From Friedrich Wöhler’s Urine to Eduard Buchner’s Alcohol

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These two pages contain the full text of the following chapter: Athel Cornish-Bowden (1997) Harden and Young’s Discovery of Fructose 1,6-Bisphosphate, pp. 135-148 in New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge (ed. A. Cornish-Bowden), Universitat de València, Valencia, Spain. A PDF file (60 kilobytes) is also available.

A road sign in southern New South Wales
(courtesy of Dr Robert Scopes)

As in the case of so many discoveries, the new phenomenon was brought to light, apparently by chance, as the result of an investigation directed towards other ends, but fortunately fell under the eye of an observer possessed of the genius which enabled him to realise its importance and give to it the true interpretation. (HARDEN, 1932)

Eduard Buchner’s discovery that a cell-free extract of yeast was capable of fermenting sugar (Buchner, 1897) opened the door to much of the development of modern biochemistry. As discussed by Bohley and Fröhlich in this book (pp. 51-60), others before Buchner, such as Marie von Mannaseïn (1897), had made similar claims, but these were based on inadequate evidence and did not lead to the explosion of new research that followed from Buchner’s work. In any investigation it is not sufficient to come to the right conclusions; one must base them on evidence firm enough to convince others. Among those who built on Buchner’s work was Arthur Harden, who devoted a large part of his career to the study of alcoholic fermentation. Harden’s assessment of Buchner’s contribution is well expressed by the sentence quoted at the beginning of this chapter. After deciding to include this quotation I was pleased to find that the same sentence had caught the eye of one of the giants of 20th century biochemistry, Fritz Lipmann (1971), who added his own comment that What I delight in here is [Harden’s] preoccupation with the accidental.

Lipmann went on to say that these remarks applied equally to Harden’s own great discovery that phosphate takes part in the chemical reactions that convert glucose to ethanol and carbon dioxide. This discovery derived, of course, from Buchner’s work, and was itself the beginning of the complete understanding of the reactions of fermentation and glycolysis. This discovery, and especially its implications for understanding the application of thermodynamic ideas to metabolism, will form the main theme of this chapter. First, however, I shall digress to discuss the importance of yeast fermentation in general, and Buchner’s work in particular, in the origins of our present ideas on enzyme kinetics in the early years of the 20th Century.

When I was reading the background literature for my first book about enzyme kinetics (Cornish-Bowden, 1976) I was working in the Department of Biochemistry at Birmingham, which had one of its roots in the British School of Malting and Brewing, and I was already conscious of the importance of brewing in the early development of biochemistry, and also of how this had been transformed by Buchner’s discovery. As early as 1892 Adrian Brown, Professor of Malting and Brewing at Birmingham, suggested that the kinetics of enzyme-catalysed reactions could be related to the occurrence of an enzyme-substrate complex along the reaction pathway (Brown, 1892), but his kinetic observations on live yeast appeared to conflict with those of O’sullivan and Tompson on isolated preparations of invertase (1890), and did not establish a new view of enzyme catalysis.

Buchner’s discovery transformed Brown’s view of his own work, and ten years later he reexamined it using purified invertase. Finding that purified invertase showed the same sort of kinetic behaviour as live yeast (Brown, 1902), he explained the now very familiar idea of enzyme saturation at high substrate concentrations in essentially the same way as we would explain it now, saying that the need to pass through an enzyme-substrate complex placed a limit on how fast a reaction could go, because this complex could not break down infinitely fast to give products.

Victor Henri (1902, 1903) criticized Brown’s ideas, not because he thought they were fundamentally wrong, but because he objected to the notion of an enzyme-substrate complex with a fixed lifetime between its abrupt creation and decay. He reformulated the hypothesis in a way that was conceptually quite similar but much more in accord with contemporary ideas of chemical kinetics. He appears to have been the first to write down an equation equivalent to what is nowadays called the Michaelis-Menten equation.

Some modern authors see it as unjust that Leonor Michaelis and Maud Menten (1913) are normally credited with discoveries that could also be attributed to Brown or Henri. In reality, however, the earlier work was open to the same criticisms that Buchner made of von Manasseïn: Brown and Henri may have been essentially right, but they had no understanding of the need to control the pH or to take account of mutarotation or effects that might alter the enzyme activity during the course of a reaction followed for an hour or more, so it is difficult to have much confidence in the experimental observations on which they based their ideas. It remained for Michaelis and Menten to define experimental standards that are not very different from those that are used today, more than 80 years later.

Incidentally, if there has been any undue neglect of Brown’s and Henri’s contributions in the subsequent development of enzymology, Michaelis and Menten cannot be blamed for it; they were careful to give proper credit to their predecessors, particularly Henri:

The investigations of Henri are especially important because starting from reasonable ideas on the nature of enzyme action he arrives at a mathematical formulation of the course of enzyme action which fits the facts quite well. In this work we also start from these ideas of Henri. We undertook to re-investigate the whole work because Henri did not take into account two influences which are of very great importance and the neglect of which in Henri’s work now appears so serious that a new investigation is worth while. The first is the effect of hydrogen ion concentration ([H⁺]), the second the effect of mutarotation. (MICHAELIS and MENTEN,1913, trans. BOYDE, 1980)

Returning now to the main subject of this chapter, the experiments of Harden and Young (1906, 1908, 1911), my interest in these was stimulated by a casual remark of Dan Koshland’s when I was making a return visit to his laboratory in the summer of 1974. Before that, I had vaguely heard of Harden and Young, and knew that the Biochemical Society sponsored a series of conferences with the title Harden Conferences, but, like most biochemists educated since 1960, especially those with a background more in chemistry than in biochemistry, I knew next to nothing of what they had achieved. (My ignorance in this matter is hardly unique: a few days before writing these words I was talking with a distinguished biochemist – in the field of carbohydrate metabolism, no less – who was shortly to attend his first Harden Conference, and he had no idea what Harden’s contribution to biochemistry had been).

Even in the 1970s not all of the standard textbooks were very illuminating, and those of today are certainly no more so. For example, White, Handler and Smith (1968) limit themselves to an index entry Harden-Young ester (see Fructose 1,6-diphosphate), which is suggestive but no more. Modern students of glycolysis can be thankful, incidentally, that the practice of naming metabolites after their discoverers has not survived, as they would have to contend not only with Harden-Young ester, but with others as well, such as Robinson ester (glucose 6-phosphate) and Neuberg ester (fructose 6-phosphate).

Lehninger (1975) is much more helpful, both because he identifies the Harden-Young experiments as the first major new steps made in the understanding of alcoholic fermentation after Buchner opened up this field, and because he makes it clear that they led to the discovery of a major metabolic intermediate, fructose 1,6-bisphosphate, and of the coenzymes NAD, ATP and ADP.

In fact, Harden and Young’s experiments were not only crucial in the development of our understanding of the glycolytic pathway; they also provide some of the most instructive examples for teaching biochemical equilibria to modern students. Dan Koshland’s remark mentioned earlier was that a thorough study of the experiments would give students more understanding of thermodynamics than a course of thermodynamics taught in the conventional way. By saying this he stimulated me to read the relevant papers and find out from them what Harden and Young had done. I thus confirmed that they do indeed provide an effective basis for teaching some of the crucial points about biochemical equilibria necessary for understanding why metabolic pathways behave as they do.

The experiments were published in a series of papers (more than the three cited here) entitled The alcoholic ferment of yeast-juice, and like most papers of their time they included much more detail than the modern reader is likely to find interesting. A more convenient, but equally authoritative source, for reading about them, therefore, is likely to be Harden’s book Alcoholic Fermentation (Harden, 1932).

As Scopes also deals with the Harden-Young experiments in his chapter in this volume (pp. 151-158), I shall concentrate here on the thermodynamic aspect that drew me to them in the first place. Understanding them is easier for us than it was for Harden and Young, because of all of the knowledge about the chemical details of glycolysis that has been gained since their time. In modern terminology, the crucial observations were as follows:

1. In the presence of an ample supply of inorganic phosphate, alcoholic fermentation of glucose by yeast extract proceeds until all of the glucose is consumed (Harden and Young, 1906).
2. Under inorganic phosphate limitation fermentation becomes very slow and fructose 1,6-bisphosphate accumulates (Harden and Young, 1908).
3. Addition of a small amount of inorganic phosphate to yeast extract fermenting under phosphate limitation causes a rapid increase in the rate of fermentation, during which 1 mol CO₂ is produced per mol phosphate added, after which very slow fermentation resumes (Harden and Young, 1908).
4. In the absence of inorganic phosphate, addition of a trace of arsenate causes a rapid increase in fermentation, which may continue until all of the glucose is consumed, and analysis shows no perceptible change in the concentration of arsenate at any time (Harden and Young, 1911).

To understand the first three of these observations it is sufficient to know the glycolytic reactions and their standard Gibbs energies *gkcapdeltaG°' shown in Table 1. To understand the fourth it is also necessary to know that glyceraldehyde 3-phosphate dehydrogenase will accept arsenate as a substrate instead of inorganic phosphate, but that the 1-arseno-3-phosphoglycerate presumed to be produced is unstable and is spontaneously hydrolysed to 3-phosphoglycerate and arsenate.

If we ignore for the moment the experiment with arsenate, it is clear that the reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase cannot proceed in the absence of inorganic phosphate and that consequently the whole process must cease. But why should fructose 1,6-bisphosphate accumulate rather than any of the other four intermediates that occur before the blocked reaction? To understand this we need to examine the *gkcapdeltaG°' values shown in Table 1.

Table 1. Thermodynamic characteristics of the early reactions of fermentation

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