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Transcript
Sir Hans Krebs and the citric acid cycle 1932 1957 1937 (1900-1981) Oxford England 1972 Sum: C2 yields 2 CO2 Glycolysis Glucose + 6 O2 6 CO2 + 6 H2O Glycolysis Glucose + 6 O2 6 CO2 + 6 H2O Glucose + 6 O2 + 32 ADP + 32 Pi 6 CO2 + 6 H2O + 32 ATP Glycolysis ADP + Pi ATP 6 O2 6 CO2 + 6 H2O Glycolysis ADP + Pi ATP 6 O2 6 CO2 Citric Acid Cycle was “discovered” by Hans Krebs • How did he do it? • What was known at the time? • What prepared him? 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). 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. 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 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 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. 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! Discovery of the urea cycle •Expert in the use of manometry. •Quick method for urea determination: Urea + H2O CO2 + 2 NH3 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. 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 First rendition of ornithine cycle Ornithine + CO2 + 2 NH3 Arginine Arginine + H2O Ornithine + Urea Sum: CO2 + 2 NH3 Urea Discovery of the urea cycle + CO2 + 2 NH3 Discovery of the urea cycle Wada – isolated citrulline from watermelons and solved its structure! Once in awhile you get LUCKY! Discovery of the urea cycle Discovery of the urea cycle 1932 2 NH3 + CO2 urea + H2O Publication of urea cycle paper 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.” 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 ………. 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. 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. Hitler’s Seizure of Power - 1933 “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 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”. The Holocaust 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 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 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 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. 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. 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. 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! 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. Glutamate 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! 2C 4C 6C Sum: 2C 2 CO2 CO2 CO2 5C What did Krebs do? Clever experiments with malonate, which inhibits succinate dehydrogenase EXPERIMENTS citrate fumarate malate oxaloacetate glucose Malonate What did Krebs do? Realized that oxaloacetate must be converted to citrate. Experimentally demonstrated that: OAA + “something derived from glucose” Citrate “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 “Detailed citric acid cycle. The information available at present about the intermediate steps may be summarized thus:” Glycolysis ADP + Pi ATP 6 O2 6 CO2 Glycolysis Fritz Lipmann – 1951 Discovered: coenzyme A; Pyruvate to acetyl-CoA Paper on the Citric Acid Cycle Submitted to Nature “The role of citric acid in intermediate metabolism in animal tissues” H.A. Krebs and W.A. Johnson Department of Pharmacology University of Sheffield Paper on the Citric Acid Cycle Timing is everything - 1939 • Krebs obtained British citizenship just a few days before the outbreak of World War II. • Spared internment in a concentration camp. May 1940: British government interned 28,000 Germans and Austrians, mostly Jewish refugees from Germany. Major Honors • Fellow of the Royal Society of London in 1947 • Nobel Prize for Medicine and Physiology in 1953 • Knighted in 1958 Moving On • 1954 University of Oxford: Whitley Chair of Biochemistry • 1967 Retired from Whitley Chair • 1967 Established the Metabolic Research Laboratory in the Radcliff Infirmary in Oxford Sense of Humor? The “Krebs” Cycle Sense of Humor? The Last Word -1981 Many obituaries were published The shortest but the best, I think, was published by the Journal Nature Thanks!