Sir Hans Krebs and the citric acid cycle

1. Sir Hans Krebs and the citric acid cycle

2. 1932 1957 1937 (1900-1981)

3. Cheers! Oxford England 1972

4. Sum: C2 yields 2 CO2

6. Glycolysis Glucose + 6 O26 CO2 + 6 H2O

8. Glycolysis Glucose + 6 O26 CO2 + 6 H2O Glucose + 6 O2 + 32 ADP + 32 Pi 6 CO2 + 6 H2O + 32 ATP

9. Glycolysis ADP + Pi ATP 6 O2 6 CO2 + 6 H2O

10. Glycolysis ADP + Pi ATP 6 O2 6 CO2

11. Citric Acid Cycle was “discovered” by Hans Krebs • How did he do it? • What was known at the time? • What prepared him?

12. The formative years of Krebs as a scientist • Born small town near Hanover - August 25, 1900 • Second child of Georg Krebs, MD, and his wife Alma. • Grammar school (1910-1918) • Served briefly in the Imperial Army near the end of World War I. • Introduced to science at University Gottingen (1919).

13. The formative years of Krebs as a scientist • Transferred to University of Freiburg where he first encountered intermediary metabolism (1919-1920) • Franz Knoop –  oxidation of fatty acids. • Transferred to Munich - graduated in Medicine with first class marks in December 1923.

14. The formative years of Krebs as a scientist • Unpaid position in a Clinic in Berlin to qualify for license to practice medicine (1924-1925). • Set up a small laboratory “on the side”. • Research assistant with Otto Warburg at the Kaiser Wilhelm Institute in Berlin • Nobel prize for Physiology and Medicine in 1931

15. Otto Warburg (1926-1930) • Technique of manometry • Tissue slice experimental system • Stoichiometry (balanced chemical reactions) • Evolution • Published 16 papers in 4 years • Warburg forced him to leave (1930) • Did not want senior scientists in his laboratory • Recommended that Krebs practice medicine as a career • Doubted Krebs could find a research position

16. The formative years of Krebs as a scientist (1930-1933) • Obtained position at Freiburg as an “Assistant” in the Department of Medicine. • Full clinical responsibility for two wards of 22 patients. First time Krebs earned a salary. • Allowed to set up a small lab with funding from the Rockefeller Foundation.

17. Discovery of the urea cycle - 1932 • Kurt Henseleit, medical student. • Krebs-Henseleit bicarbonate buffer • Humans produce 30 grams of urea from protein in 24 h • Liver slices • Quantitative measurements and stoichiometry • Manometry!

18. Discovery of the urea cycle • Expert in the use of manometry. • Quick method for urea determination: Urea + H2O CO2 + 2 NH3

19. Discovery of the urea cycle • Liver rich in arginase: Arginine + H2O ornithine + urea • Might be part of a general urea-synthesizing mechanism. • More arginase present in the liver than needed for degradation of arginine. • The principle of evolution.

20. Discovery of the urea cycle - 1932 • Synthesis of urea from ammonia greatly increased by ornithine. • One molecule of ornithine caused formation of many molecules of urea. • Sub-stoichiometry! Not used up in the reaction! A catalyst! • A catalyst takes part in a reaction and forms an intermediate but must be regenerated. • Suggested ornithine is converted to something which is converted back to ornithine with formation of urea. • Something = arginine

21. First rendition of ornithine cycle Ornithine + CO2 + 2 NH3 Arginine Arginine + H2O Ornithine + Urea Sum: CO2 + 2 NH3 Urea

22. Discovery of the urea cycle + CO2 + 2 NH3

23. Discovery of the urea cycle Wada – isolated citrulline from watermelons and solved its structure! Once in awhile you get LUCKY!

24. Discovery of the urea cycle

25. Discovery of the urea cycle - 1932 2 NH3 + CO2 urea + H2O

26. Publication of urea cycle paper

27. Ornithine (Urea) Cycle • A new pattern in the organization of metabolic processes • 100’s of “metabolic” cycles are now known • Immediate and world-wide recognition of Krebs! • Good timing. “By good fortune, I published my work on the ornithine cycle just a few months before Hitler came to power.”

28. Hitler’s Seizure of Power - 1933 • Scientific success did not protect Krebs against anti-Semitism. • Hitler elected German Chancellor in January of 1933. • Two months later ……….

29. Order of the National Socialist Government On the Maintenance of Security and Order The Minister of the Interior has decided that all members of the Jewish race (irrespective of their religion) who are employed in the service of the State or in teaching establishments will be placed on leave of absence until further notice.

30. Letter from the Director of the Hospital I am instructed by the Minister of Education to inform you that you have been relieved of your post in connection with the Law for the Reconstruction of the Professional Civil Service. Your contract will terminate on 1 July 1933.

31. “If the dismissal of Jewish scientists means the annihilation of contemporary German science, then we shall do without science for a few years.” - Adolf Hitler Hitler’s Seizure of Power - 1933

32. History of Jews in Nazi Germany • 1933 - Persecution of Jews by Nazis began. • 1935 - Law enacted for the “Protection of German Blood and Honor”. • 1939 - World War II started. • 1940 - The Holocaust began; Jews sent to concentration camps; 6 million European Jews murdered. • 1943 – Germany declared “clean of Jews”.

33. The Holocaust

34. Cambridge (1933-1935) • Warburg – “I could offer you a post - but England seems safer for you. We do not know what might happen here.” • Nazis barred Krebs from entering his laboratory • Obtained temporary position at Cambridge in England • 16 wooden boxes and several suitcases - two complete Warburg baths, 24 manometers, and many manometric flasks • Moved to Cambridge in June 1933

35. Sheffield (1935) • Two years at Cambridge. • Felt insecure as a refugee from Germany in a temporary position • Lectureship in Pharmacology at the University of Sheffield in 1935

36. Citric Acid Cycle - 1937 Were there things that prepared/enabled him to do it? • Evolution • Manometry • Accurate, quantitative measurements • Stoichiometry! • Discovery of the urea cycle • Think in terms of the big picture. • A good graduate student: William A. (Johnny) Johnson

37. What was “known” at the time he discovered the citric acid cycle? • Foodstuffs are “combusted”, i.e. burned in the presence of oxygen to carbon dioxide and water. • Glucose + 6 O2 6 CO2 + 6 H2O • Glucose “fermented” to pyruvate and lactate in animal tissues and to pyruvate and ethanol in yeast.

38. Why were the steps of fermentation worked out before the citric acid cycle? • Fermentation by yeast yields ethanol, essential ingredient of “good brews”. • Glycolytic enzymes are soluble and stable in cell-free extracts. • Citric acid cycle enzymes are located in “insoluble cell structures” and are either not present or not stable in cell-free extracts.

39. Albert Szent-Gyorgyi • Hungarian-American biochemist. • Coarsely minced breast muscle of the pigeon – intact mitochondria. • Believed intermediates involved in oxidation of foodstuffs could be identified by measuring their rates of oxidation. • Established that succinate, fumarate, malate, and oxaloacetate were burned at high rates.

41. Albert Szent-Gyorgyi Most important discovery! Rate and amount of oxygen consumption is greatly increased by adding small amounts of succinate, fumarate, malate, and oxaloacetate. Sub-stoichiometry! Catalytic! But could not see what this suggested!

42. Franz Knoop and Carl Martius • German biochemists • Not concerned with glucose oxidation • Oxidation of citric acid in orange juice! • Established that citric acid is converted aconitic acid which is converted to isocitric acid which is converted to -ketoglutarate.

43. Glutamate

44. What did Krebs do? • Quantitated the rates of oxidation of tricarboxylic and dicarboxylic acids • Quantitated the catalytic effects of the tricarboxylic and dicarboxylic acids. Suggested a cycle!

45. 2C 4C 6C Sum: 2C 2 CO2 CO2 CO2 5C

46. What did Krebs do? Clever experiments with malonate, which inhibits succinate dehydrogenase

47. EXPERIMENTS citrate fumarate malate oxaloacetate glucose Malonate

48. What did Krebs do? Realized that oxaloacetate must be converted to citrate. Experimentally demonstrated that: OAA + “something derived from glucose” Citrate

50. “These facts suggest that citric acid acts as a catalyst in the oxidation of carbohydrate in the following manner:” Two enormous contributions: Conceptual: Must be dealing with a cycle. Experimental: conversion of OAA to citric acid