Fritz Lipmann

Not enough of a crook for the law

Fritz Albert Lipmann was born on 12 June 1899 in Königsberg, the capital of East Prussia: a Hanseatic port near the Russian border that is now Kaliningrad. He came from a Jewish family; his father, Leopold, was a lawyer, his mother was Gertrud, née Lachmanski. By his biographers’ account he was an unremarkable pupil who preferred Latin to Greek, and his father told him he ‘was not enough of a crook to be an outstanding lawyer.’ The death of an uncle he idolised, from a ruptured appendix, helped push him toward medicine, which he began in Munich in 1917.

In 1918 he was called up into the army medical service near the front; back in Königsberg after the war he worked through the influenza epidemic for months without catching it. In 1919 he rejoined his brother Heinz in Munich, where the brothers spent much of their time among the painters, writers and theatre people of Schwabing. The painter Friedel Sebba later portrayed the two of them together (1926). Lipmann’s medical studies took him between Königsberg, Berlin and Munich until 1922.

Medicine did not hold him. Three months of dissecting cadavers and reading slides in pathology turned him away from practice; a three-month biochemistry course in Berlin taught by Peter Rona, a former co-worker of Leonor Michaelis, turned him toward the laboratory. Lipmann called it a ‘biochemical marathon’. Rona’s students also included Hans Krebs and Ernst Chain. In 1923, with hyperinflation wrecking Germany, he took a fellowship in Ernst Laqueur’s pharmacology department in Amsterdam and decided there that he would be a biochemist, and that he had to learn chemistry properly. His Berlin MD (1924) rested on a colloid-chemistry paper with Rona; he then studied organic chemistry under Hans Meerwein.

Creatine phosphate in Meyerhof’s Dahlem

From 1926, supported at first by his father, he worked in Otto Meyerhof’s laboratory at the Kaiser Wilhelm Institute in Berlin-Dahlem. The building was crowded with future history: Karl Lohmann, who discovered ATP there, Severo Ochoa, Dean Burk and David Nachmansohn worked with Meyerhof, while Otto Warburg, in whose laboratory Hans Krebs worked, sat on the top floor and seldom came down. Lipmann never worked for Warburg, but in his memoirists’ phrase Warburg’s influence ‘permeated the institute’. Decades later, when François Chapeville mentioned over lunch that he had worked with Lipmann, Warburg replied, ‘Ah, then you are my grandson.’

The question in the building was where muscle gets its energy. A. V. Hill and Meyerhof had tied contraction to the lactic acid made by glycolysis. Then Einar Lundsgaard in Copenhagen blocked glycolysis with iodoacetate: muscles still contracted, and injected rats went into rigor and died within minutes. The energy was coming from the breakdown of creatine phosphate. When Lundsgaard joined Meyerhof’s group, Lipmann stimulated muscle through platinum electrodes sealed into manometer vessels and showed a quantitative relation between creatine phosphate breakdown and contraction. Creatine phosphate turned out to be a reservoir of phosphate groups that could be fed into ATP: the seed of the idea Lipmann generalised a decade later.

He also worked out how fluoride poisons muscle: it blocks glycolysis and binds methaemoglobin. Accounts of his degrees differ. The Nobel Foundation’s biography gives a Berlin PhD in 1927 and has him following Meyerhof to Heidelberg; the Royal Society memoir by William Jencks and Richard Wolfenden gives a 1927 chemistry doctorate with Meerwein and a 1929 PhD sponsored by Carl Neuberg. In 1929, at the Berlin Sozialistenball, he met Freda Hall, an Ohio-born fashion illustrator. Theatre was family territory, since Heinz worked with the director Leopold Jessner at the Staatstheater, and Lipmann later described theatre people as a ‘clan, closed up and agitated by their problems and intrigues’, full of ‘unusual characters and beautiful women, often astonishingly intelligent, who were not unsimilar to scientists.’ When the Meyerhof fellowship could not be renewed, he found his first paid job with difficulty, in 1930, in Albert Fischer’s tissue-culture laboratory in Berlin, measuring the growth of fibroblasts by their oxygen uptake.

Serine phosphate, Copenhagen and a change of buffer

He married Freda Hall on 23 June 1931 and left on a Rockefeller Foundation fellowship for P. A. Levene’s laboratory at the Rockefeller Institute in New York. He went looking for an energy-rich phosphate–nitrogen link in phosphoproteins, like the one in creatine phosphate. Instead he isolated serine phosphate from egg-yolk protein, a phosphate ester so stable that it survived strong acid. Why so much protein in brain and other tissues carried phosphate on serine stayed a mystery until hormone signalling was traced to enzymes that phosphorylate proteins; Lipmann later regretted having stumbled on that gold mine and walked past it. In the summer of 1932 he worked at Woods Hole in Leonor Michaelis’s rooms.

Fischer had meanwhile been given a new institute in Copenhagen, funded by the Rockefeller and Carlsberg foundations, and in 1932 Lipmann followed him as research associate at the Carlsberg Foundation’s Biological Institute. Hitler came to power in Germany the next year; Lipmann stayed in Denmark. He began on the Pasteur effect, the way yeast and tissues stop wasteful fermentation when given air, and failed to find any direct effect of oxygen on the glycolytic enzymes. The regulator turned out, much later, to be the cell’s ratio of ATP to ADP.

From 1937 he studied pyruvate oxidation in a strain of Lactobacillus delbrueckii, because pyruvate is where respiration branches off from fermentation. The decisive observation, in his own account, was accidental. To measure methylene blue reduction manometrically he swapped his usual phosphate buffer for bicarbonate, and pyruvate oxidation nearly stopped; a little phosphate brought it back. When he added adenylic acid, inorganic phosphate disappeared into ATP. Oxidation, not only glycolysis, was making ATP, and he concluded that the missing intermediate was acetyl phosphate. He presented the work at the 1939 Cold Spring Harbor Symposium.

With fascism and antisemitism rising in Germany, Danish friends warned him it was dangerous to stay. Dean Burk, his old colleague from Meyerhof’s laboratory, was moving to Vincent du Vigneaud’s department at Cornell Medical School in New York; with Kaj Linderstrøm-Lang’s recommendation, du Vigneaud took Lipmann on in 1939. There, with Rollin Hotchkiss next door at the Rockefeller Institute, he showed that the bacterial antibiotics tyrocidine and gramicidin contain unusual D-amino acids. He would come back to them decades later.

The squiggle

In the spring of 1940, on holiday in Vermont, Lipmann thought through what acetyl phosphate, creatine phosphate and ATP had in common. The result was ‘Metabolic generation and utilization of phosphate bond energy’, the long review he wrote for the first volume of Advances in Enzymology in 1941. It introduced the squiggle, ~P, for ‘energy-rich’ phosphate.

The idea, plainly put: not every phosphate attached to a molecule is equal. Split the phosphate off an ordinary sugar phosphate and little free energy is released. Split it from creatine phosphate, from the acyl phosphate 1,3-diphosphoglycerate, from phosphoenolpyruvate or from the outer phosphates of ATP, and a great deal is released. Lipmann marked the second kind with a squiggle. In this picture glycolysis and respiration load phosphate into squiggle positions, ATP collects them, and muscle contraction, transport across membranes and the building of proteins and nucleic acids spend them. In his Nobel lecture he described proposing ‘the phosphate bond as a versatile energy distributing system’ and, beyond that, ‘a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis’: the programme the rest of his career worked through.

The squiggle is bookkeeping rather than literal chemistry. What it marks is a large, favourable free energy for the whole hydrolysis reaction, not energy packed inside a single bond. As bookkeeping it worked, and it remains the way coupling is taught.

Being right on paper did not get him a job. At a big 1940 symposium on intermediary metabolism in Madison, Wisconsin, he was asked to speak on the Pasteur effect, which no longer interested him. His lecture was far too long, and the chairman, Carl Neuberg, stopped him halfway through. Jencks and Wolfenden write that word of the fiasco hurt his search for a post. What came, in 1941, was a Ciba fellowship in Edward Churchill’s Department of Surgery at the Massachusetts General Hospital in Boston, paid for by a grant obtained by the surgeon Oliver Cope: a biochemist parked in a surgery department.

The acetylation problem

At Mass General the 1941 paper quickly drew biochemists to his laboratory, the Commonwealth Fund paid for the work, and Constance Tuttle became his technician for many years. Lipmann wanted to test whether acetyl phosphate was the ‘active acetate’ that isotope experiments showed feeding into fatty acids, sterols and amino acids. He needed an easy assay. Liver acetylates sulphonamide drugs, and the product can be measured by colour. Rabbit liver homogenate did it weakly; pigeon liver did it powerfully.

The test failed instructively. Animal tissues are full of an enzyme that destroys acetyl phosphate within minutes, and even massive doses of it supplied no acetyl groups, while ATP plus acetate worked. Then the pigeon-liver extract began to die: left for four hours at room temperature, or dialysed, it lost its power to acetylate. Boiled liver extract, in which the enzymes are destroyed, brought it back. Something small and heat-stable was being used up or washed out. It was present in boiled extracts of every organ, in microbes and in yeast, and no known cofactor could replace it.

In a two-page note in 1946, Lipmann and Nathan Kaplan reported that the same factor drove the acetylation of choline in brain extracts, matching the heat-stable factor found by David Nachmansohn’s group and by Wilhelm Feldberg and Mann. Lipmann named it coenzyme A, the A for the activation of acetate. Kaplan and G. David Novelli purified it from pig liver. In Roger Williams’s laboratory, Beverly Guirard surveyed a 40 per cent pure preparation for vitamins and at first found none; noticing that the pantothenic acid reading crept up with long enzyme digestion, she hydrolysed the coenzyme with acid and found about 11 per cent pantothenic acid. The 1947 paper by Lipmann, Kaplan, Novelli, Tuttle and Guirard announced a coenzyme built from a B vitamin; it later emerged that essentially all the pantothenic acid in a cell is in CoA.

Taking the coenzyme apart

Working out the structure took six years and several laboratories. Two enzymes, intestinal phosphatase and something in pigeon-liver extract, each inactivated CoA and together released pantothenic acid, implying two attachment points. The phosphate side proved to be a pyrophosphate bridge to adenylic acid, identified by Novelli enzymatically and by J. Baddiley chemically. Novelli also found a third phosphate, which Kaplan placed on the 3′ position of the ribose with a phosphatase from rye grass that cuts only there. John Gregory, working with the Upjohn Company, supplied a nearly pure preparation from Streptomyces fradiae. On the other side, E. E. Snell’s group showed that the sulphur-bearing piece was thioethanolamine joined to pantothenic acid by a peptide bond. Baddiley, E. M. Thain, Novelli and Lipmann published the structure in Nature in January 1953.

The sulphur was the point. Feodor Lynen isolated acetyl-CoA (1951) and showed that the acetyl group sits on CoA’s sulphur as a thioester, an energy-rich link like acetyl phosphate; Lipmann’s lecture calls it ‘the brilliant work of Feodor Lynen’. Acetyl-CoA was the active acetate: CoA carries two-carbon pieces the way ATP carries phosphate. Morris Soodak in Lipmann’s laboratory showed CoA was needed to make acetoacetate; Joseph Stern and Severo Ochoa showed CoA-dependent citrate synthesis, acetyl-CoA joining oxaloacetate at the entry to the citric acid cycle. Mary Ellen Jones, with Lipmann, Helmuth Hilz and Lynen, found the surprising way animals make acetyl-CoA: ATP is split into AMP and pyrophosphate.

In 1949 Lipmann became professor of biological chemistry at Harvard Medical School while running his group in the hospital’s Biochemical Research Laboratory, where Novelli, Kaplan and Jones acted as senior hands for the newcomers. In 1953 he shared the Nobel Prize in Physiology or Medicine with Hans Krebs: Lipmann for coenzyme A and its importance in intermediary metabolism, Krebs for the citric acid cycle. Acetyl-CoA is what feeds Krebs’s cycle, so the two halves fit. His Nobel lecture is candid about the disappointments along the way, acetyl phosphate failing in animal tissue, and admits that the excitement of finding the vitamin had ‘blinded us for a long time to other possibilities.’