Classic Articles

Otto Fritz Meyerhof and the Elucidation of the Glycolytic Pathway

The Equilibria of Isomerase and Aldolase, and the Problem of the Phosphorylation of Glyceraldehyde Phosphate (Meyerhof, O., and Junowicz-Kocholaty, R. (1943) J. Biol. Chem. 149, 71-92)

The Origin of the Reaction of Harden and Young in Cell-free Alcoholic Fermentation (Meyerhof, O. (1945) J. Biol. Chem. 157, 105-120)

The Mechanism of the Oxidative Reaction in Fermentation (Meyerhof, O. and Oesper, P. (1947) J. Biol. Chem. 170, 1-22)

The elucidation of the glycolytic pathway, the process whereby glucose is converted into pyruvate and ATP, began in 1860 when Louis Pasteur observed that microorganisms were responsible for fermentation. Several years later, in 1897, Eduard Buchner made the significant discovery that cell-free extracts could carry out fermentation. The next important contribution was from Arthur Harden and William Young in 1905. They realized that inorganic phosphate was necessary for glycolysis and that fermentation requires the presence of both a heat-labile component they called “zymase” and a low molecular weight, heat-stable fraction called “cozymase.” (It was later shown that zymase contains a number of enzymes whereas cozymase consists of metal ions, ATP, ADP, and coenzymes such as NAD.) Building on these initial observations, the complete glycolytic pathway was elucidated by 1940 by the combined efforts of several scientists including Otto Fritz Meyerhof (1884-1951).

Meyerhof was born in Hanover, Germany and grew up in Berlin. In 1909, he graduated as a doctor of medicine from the University of Heidelberg. Around this time, Ludolf von Krehl was building a small research program on metabolism at the University of Heidelberg Medical Clinic, and he offered Meyerhof a position in his laboratory. There, Meyerhof met Otto Warburg whose innovative ideas and confident approach inspired him to focus his career on physiological chemistry.1

In 1912, Meyerhof took a position at the University of Kiel. A year later, he delivered a lecture on the energetics of living cells, one of the very first adaptations of the physical laws of thermodynamics to physiological chemistry. Meyerhof had recognized that after energy is input as food it is transformed through a series of intermediate steps and finally dissipated as heat. He soon began using muscle to look at energy transformations and chemical changes during cellular function. Meyerhof was also interested in analogies between oxygen respiration in muscle and alcoholic fermentation in yeast and proved, in 1918, that the coenzymes involved in lactic acid production were the same as the yeast coenzymes discovered by Harden and Young, revealing an underlying unity in biochemistry.

Soon after World War I, Meyerhof began collaborating with Archibald Vivian Hill who was investigating heat production in muscle. The pair worked to decipher metabolism in terms of heat development, mechanical work, and cellular chemical reactions. Meyerhof determined that glycogen is converted to lactic acid in the absence of oxygen and showed that in the presence of oxygen only a small portion of lactic acid is oxidized and the rest is converted back to glycogen. This discovery of the lactic acid cycle provided the first evidence of the cyclical nature of energy transformation in cells. These results also confirmed and extended Louis Pasteur's theory (now called the Pasteur-Meyerhof effect) that less glycogen is consumed in muscle metabolism in the presence of oxygen than in its absence. Meyerhof and Hill won the Nobel Prize in Physiology or Medicine in 1922 for their analysis of the lactic acid cycle and its relation to respiration.

Otto F. Meyerhof. Photo courtesy of the National Library of Medicine.

Two years after wining the Nobel Prize, Meyerhof joined the Kaiser Wilhelm Institutes in Berlin-Dahlem. Then, in 1929, he took charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.

By this time, it was clear that glycolysis was far more complicated than anyone had imagined. The sheer number of components and their short lived nature made the task of sorting out the pathway daunting. However, during his time at Heidelberg, Meyerhof's group was extremely successful at breaking down glycolysis into its many separate components. In 1932, Meyerhof made the first associations between the uptake of phosphate during the breakdown of carbohydrates to lactic acid and the splitting of ATP. By 1934, Kurt Lohmann in Meyerhof's laboratory provided direct evidence that ATP synthesis was the byproduct of utilization of glucose. Lohmann also established that creatine phosphate is an energy source for ATP phosphorylation, which led Meyerh of to the conclusion that the energy release from ATP hydrolysis was the primary event leading to muscle contraction.

By the 1930s Meyerhof had managed to isolate and purify the co-enzymes involved in the conversion of glycogen to lactic acid and had reconstructed the main steps of this set of reactions in cell-free solution. All in all, Meyerhof's group discovered more than one-third of the enzymes involved in glycolysis. In 1932, Gustav Embden constructed a detailed proposal for reaction sequences for almost the entire glycolytic pathway. Over the next 5 years, Meyerhof, along with Warburg, Jacob Parnas, Carl Neuberg, Gerti and Karl Cori, and Hans von Euler worked out the details of glycolysis, which is often referred to as the Embden-Meyerhof pathway.

With Adolf Hitler's rise to power, Meyerhof left Germany in 1938 and became director of the Institut de Biologie Physiochimique in Paris. In 1940, when the Nazis invaded France, Meyerhof fled to the United States where the post of Research Professor of Physiological Chemistry was created for him by the University of Pennsylvania and the Rockefeller Foundation. He remained at Pennsylvania where he continued to study metabolism until his death. The three Journal of Biological Chemistry (JBC) Classics reprinted here are from Meyerhof's time at Pennsylvania.

The first paper deals with one of the intermediate reactions that occurs in glycolysis: the splitting of hexose diphosphate (now known as fructose 1,6-bisphosphate) into two triose phosphate isomers, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, by zymo-hexase (fructose-1,6-bisphosphate aldolase). Triose-phosphate isomerase then converts dihydroxyacetone phosphate into glyceraldehyde 3-phosphate. In the next step of glycolysis, glyceraldehyde 3-phosphate is oxidized and phosphorylated to become 1,3-diphosphoglyceric acid. Warburg and Christian (1, 2) and Negelein and Brömel (3, 4) proposed that this step occurs through the intermediate 1,3-diphosphoglyceraldehyde with the aid of an oxidizing enzyme and cozymase. If this were true, then inorganic phosphate could be used to remove glyceraldehyde 3-phosphate from the hexose diphosphate reaction.

To investigate this matter further, Meyerhof and Renate Junowicz-Kocholaty redetermined the equilibrium constant for the isomerase and aldolase reactions in the presence and absence of inorganic phosphate, cozymase, and Warburg's oxidizing enzyme. They found that their values agreed with those previously determined and that equilibrium is not influenced by the presence of inorganic phosphate, cozymase, or Warburg's enzyme. They were also unable to detect the formation of any substance that would break down into glyceraldehyde phosphate and phosphate, prompting them to write that Warburg's claims of a diphosphoglyceraldehyde intermediate may have been “premature.”

The second Classic deals with the next two steps of glycolysis shown as Reactions 1 and 2. Formula Formula

Harden and Young stated that during fermentation, one sugar molecule is fermented to CO2 and alcohol while a second is esterfied to hexose diphosphate (5). In a cell-free system, this reaction can be divided into two phases, a rapid “phosphate period” and a slower phase that depends on the rate of hexose diphosphate fermentation. Meyerhof proposed that the rate of the hexose diphosphate reaction was much slower in cell-free systems than in live yeast because the majority of the enzyme needed to split ATP, adenylpyrophosphatase (apyrase), was lost during the extraction process. He backed up his claim by studying the distribution of apyrase in the yeast cell and showing that it remains mainly with solid elements that are not used in cell-free systems. Meyerhof also purified apyrase from potatoes and added it to cell-free preparations to prove that it raises the rate of hexose diphosphate fermentation.

The final JBC Classic revisits the phosphorylation of glyceraldehyde 3-phosphate and its subsequent oxidation. In this paper, Meyerhof and Peter Oesper use a Beckman spectrophotometer to follow the reaction and provide further proof that a diphosphoglyceric aldehyde intermediate does not exist. They also alter the equation for this step of glycolysis to reflect the fact that the reduction of cozymase is accompanied by the formation of an H+ ion.

Footnotes

  • 1 All biographical information on Otto Fritz Meyerhof was taken from Ref. 6.

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