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Luminaries Submitted 12.30.09 | Revision Received 1.27.10 | Accepted 2.2.10 Sir Hans Adolf Krebs: Architect of Metabolic Cycles Bryan A. Wilson,1 Jonathan C. Schisler,2 Monte S. Willis, MD, PhD2 (1Wake Forest University, Winston-Salem, 2University of North Carolina, Chapel Hill, NC) DOI: 10.1309/LMZ5ZLAC85GFMGHU Sir Hans Adolf Krebs (1900–1981) Sir Hans Adolf Krebs was born August 25, 1900, at the dawn of a new century in Hildesheim, Germany. He was the son of Dr. Georg Krebs, an ear, nose, and throat surgeon, and his wife Alma Davidson. As a young child, Krebs attended Lutheran schools, despite his Jewish heritage; his parents rarely even mentioned Judaism in the household. Hans Krebs went on to study medicine at the Universities of Göttingen, Freiburg-im-Breisgau, and Berlin. In 1925, he earned his MD degree at the University of Munich. Following his medical education, Dr. Krebs spent an additional year studying chemistry in Berlin. In 1926, he was appointed assistant to Professor Otto Warburg at the Kaiser Wilhelm Institute for Biology. He studied under Dr. Otto Warburg for 4 years before returning to clinical work. Later in his career, Dr. Krebs shared his sincere gratitude for Dr. Warburg’s mentoring and training in research. In Dr. Warburg’s laboratory, he learned manometry to measure oxygen consumption of tissue slices, which allowed the precise investigation of biochemical (metabolic) pathways from animal tissues. These techniques were essential tools Dr. Krebs later used to discover the citric acid cycle and other novel metabolic pathways. Discovery of the Ornithine Cycle Between 1930 and 1933, Dr. Krebs practiced medicine at the Municipal Hospital at Altona under Professor L. Lichtwitz as well as Professor S. J. Thannhauser at the Medical Clinic of the University of Freiburg-im-Breisgau. In addition to his clinical duties, Dr. Krebs embarked on research in metabolism. One of the waste products the body must rid itself of is nitrogen, which it does by making and excreting urea. At the time it was known that urea production occurred in the liver, however the underlying pathways involved in urea metabolism were not defined. Without urea metabolism, the body does not have a way to get rid of nitrogenous waste, such as ammonia, which can lead to encephalopathy (Insert 1). This occurs commonly in patients with severe liver failure, where the Corresponding Author Monte S. Willis, MD, PhD [email protected] labmedicine.com ability to metabolize urea is compromised. Dr. Krebs applied his knowledge of tissue slice analysis to delineate urea metabolism. He observed urea production through the addition of the amino acid ornithine in the presence of ammonia.1 It was known since 1904 that arginine could be hydrolyzed by the Encephalopathy: A syndrome of global brain dysfunction. It manifests as an altered mental state, including involuntary muscle twitching, abrupt loss of muscle tone, seizures, etc. The underlying causes may be infectious (bacteria, virus, prion), due to metabolic/mitochondrial dysfunction, brain tumor, exposure to toxins, radiation, trauma, poor nutrition, or lack of oxygen to name a few. enzyme arginase to form ornithine and urea,1 so Dr. Krebs also explored methods in which he could synthesize arginine. Using his liver tissue slice assay with purified ornithine and citrulline, which he hypothesized was an intermediate of arginine, Dr. Krebs observed that citrulline acted as a catalyst to promote the production of urea from ammonia and carbon dioxide, thus generating the data that led to the discovery of the ornithine cycle with Kurt Henseleit in 1932.1 Around this time the rise of the National Socialist Government in Germany resulted in personal repercussions for Dr. Krebs. The political pressures of Hitler influenced all of Germany, resulting in the dismissal of all members of the Jewish race, irrespective of their religious views, employed at teaching universities (Insert 2). The National Socialist party influenced the entire German empire and many great scientists aside from Dr. Krebs fled the region. Dr. Krebs left for England in 1933 when the Nazi party took power, and he remained there until the end of his career. “Our national policies will not be revoked or modified, even for scientists. If the dismissal of Jewish scientists means the annihilation of contemporary German science, then we shall do without science for a few years.” — Adolph Hitler June 2010 ■ Volume 41 Number 6 ■ LABMEDICINE 377 Luminaries Dr. Krebs accepted a Rockefeller studentship invitation from Sir Frederick Gowland Hopkins to the School of Biochemistry in Cambridge, England, where he was quickly appointed to the position of demonstrator of biochemistry in 1934. The following year Dr. Krebs was appointed as lecturer in pharmacology at the University of Sheffield where he quickly moved up the ranks and became lecturer-in-charge of the Department of Biochemistry in 1938. This same year Dr. Krebs married Margaret Cicely Fieldhouse of Wickersley, Yorkshire, and went on to have 2 sons, Paul and John, and 1 daughter, Helen. The Tricarboxylic Acid Cycle Discovered At the University of Sheffield, Dr. Krebs and William Johnson published the work that led to the discovery of the citric acid cycle (Figure 1A).2,3 These studies were performed in the pigeon breast muscle, which is the powerful muscle necessary for flight. This was a particularly good model as this muscle maintained its oxidative capacity after its disruption and suspension in aqueous media. In these studies, Dr. A B Krebs noticed muscle tissue took up oxygen rather quickly, especially in the presence of pyruvate or lactic acid. Believing that muscle cells could not carry out the metabolism of carbohydrates in 1 step alone, he hypothesized that carbohydrate metabolism occurred in a series of defined steps, extracting the biochemical energy of carbon-based nutrients into usable cellular energy. Some of the clues to this were published by Szent–Györgyi, who demonstrated succinate, fumarate, malate, and oxaloacetate (all 4-carbon, C4, acid salts) oxidized as quickly as pyruvate and lactic acid (3-carbon acid salts) and catalytically promoted oxygen uptake.4 Another piece of the puzzle, published by Dr. Krebs in 1937, showed succinate could be synthesized by animal tissues in the presence of pyruvate.2 Dr. Krebs speculated that the 4-carbon acid salts may have been derived through the oxidation of citrate. In 1937, Martius and Knoop reported the fate of citrate undergoing oxidation.5,6 They identified that α-ketoglutarate was formed when citrate was oxidized using liver and cucumber seeds and suggested cis-aconitate and isocitrate were intermediates.5,6 Wagner-Jauregg and Rauen identified that isocitrate behaved similarly in cucumber seed extracts.7 This led Dr. Krebs to question whether the 4 carbon acid salts were in fact derived from citrate and prompted further investigation into the properties of this 6-carbon compound. Dr. Krebs observed a rapid oxidation of citrate, but interestingly, he identified that citrate was never fully consumed as a substrate, suggesting a capacity for citrate synthesis in this system (Figure 1). In addition, hypoxic conditions (low oxygen) resulted in large amounts of citrate formation in minced muscle only in the presence of both oxaloacetate and pyruvate. Two other key findings from these studies were: (1) succinate is more reduced than oxaloacetate; and (2) succinate formed from oxaloacetate coincided with a rapid uptake of oxygen.2,3 Taken together these data described a cyclic sequence of reactions, which Dr. Krebs and Johnson called the citric acid cycle (Figure 1A). Figure 1_The citric acid “Krebs” cycle: Then and now. (A) The citric acid cycle as proposed (and referenced) by Krebs and Johnson in 1937. (B) The chemical intermediates are in bold and the enzymes responsible for driving the cycles are in italics. Defects in the enzymes shown in red are responsible for known enzymopathies. Adapted from: Krebs and Johnson, 193722 and Munnich, 2008.12 378 LABMEDICINE ■ Volume 41 Number 6 ■ June 2010 The Discovery of the Glyoxylate Cycle In 1945, Dr. Krebs’ appointment at the University of Sheffield was raised to that of professor and director of a Medical Research Council’s (MRC) research unit within his department. In 1954, following years of hard work at the University of Sheffield, Dr. Krebs was appointed Whitley Professor of Biochemistry at the University of Oxford. The MRC Unit for Research in Cell Metabolism then transferred to Oxford with him. Dr. Krebs spent many years conducting confirmatory experiments for his proposed citric acid cycle. The discovery of acetyl-coenzyme A (AcetylCoA) helped to solidify Dr. Krebs proposed cycle (Figure 1B). However, the citric acid cycle failed to answer questions on how organisms could survive and grow on acetate alone to build carbon skeletons. The answer to this was elucidated in 2 key reactions with the help labmedicine.com Luminaries Table 1_Gene Mutations Identified in Citric Acid Cycle Enzymes (Figure 1) Underlying Inborn Errors of Metabolism and Disease Enzyme Affected (Gene Name) Disease Phenotype Explanation Isocitrate dehydrogenase (IDH3B) Retinitis pigmentosa13 Abnormalities of eye photoreceptors leading to progressive visual loss a-keoglutarate dehydrogenase (OGDH) Encephalopathy14 Degenerative (worsening) brain injury due to disease Succinyl-CoA synthase (SUCLA2, SUCLG1) Encephalopathy15,16 (see above) 17 Succinate dehydrogenase (SDHA, SDHB, SDHC, SSHS) Leigh syndrome Metabolic disorder of neurons in the brain leading to encephalopathy Paraganglioma18-20 Tumor/cancer of brain neurons controlling endocrine (hormone) function pheochromocytoma18,20 Tumor/cancer of adrenal glands (neuron/endocrine (hormone) connection) Fumarase (FH) Seizures Brain neuron dysfunction Muscle weakness Muscle dysfunction Encephalopathy21 (see above) Leiomyomatosis16 Tumor/cancer of smooth muscle of the uterus Adapted from Hartong and colleagues, 2008.13 of Dr. Krebs’ previous mentor, Hans Kornberg. Kornberg and Dr. Krebs characterized the actions of 2 enzymes, malate synthase, which condenses acetate with glyoxylate to form malate,8 and isocitrate lyase which provides glyoxylate for the reaction by cleaving it from isocitrate.8 These reactions bypass 2 reactions of the citric acid cycle and were originally called the glyoxylate bypass of the citric acid cycle, now referred to as the glyoxylate cycle.8,9 The citric acid cycle brought Dr. Krebs international fame, and it is considered to this day his greatest scientific achievement. Fritz Lipmann has called this work a superb survey, which is “still today the most comprehensive discussion of the central issues in the analysis of the inner workings of the living organism and highly recommended reading to beginners in biochemistry.”10 Dr. Krebs received numerous awards for his work in metabolic cycling. In 1953, he was awarded the Nobel Prize for Medicine and Physiology, jointly with Fritz Lipmann. His fame and accolades attracted attention to his department as a major center for biochemistry research, recruiting students from all parts of the world. Shortly after winning the Nobel Prize, Dr. Krebs was knighted in 1958, which changed his official title to Sir Hans Adolf Krebs. As important as his great scientific achievements, Dr. Krebs served as a great teacher, evidenced by the success of his students; several of whom went on to become well-known independent researchers across the world. In his autobiography, Dr. Krebs states with pride the success he had as an academic mentor and teacher.10 He described his success as luck but reminded us that “chance favors prepared minds.”11 He firmly believed that his success was due to training, especially his mentoring under Dr. Otto Warburg. Dr. Krebs made mentoring students just as important as science, with the hope of emulating his mentor, Otto Warburg. Our understanding of the Krebs cycle has been crucial to our understanding of health and disease on a number of levels. It describes how cells convert sugars, fats, and proteins into intermediates to supply crucial energy (ATP) essential for the support of life. A practical understanding of the importance of the citric acid cycle can be seen in inborns’ errors of metabolism, where this cycle is disrupted.12 Table 1 demonstrates the symptoms and various diseases lacking enzymes labmedicine.com essential for intermediary metabolism of the citric acid cycle (Figure 1B). While each of the enzymes of the citric acid cycle have previously been thought to be essential for life, alternative pathways certainly exist, and these pathways may be tissue specific.12,13 The details of the Krebs cycle in health and disease are still being elucidated.12,13 Dr. Krebs will be remembered for his great contributions to science and medicine. His novel methods of exploring the metabolic processes taking place in living organisms provide the fundamental basis for understanding inborn errors of metabolism. His work resonates in many fields, including metabolism, biochemistry, and medicine. LM 1. Kossel A, Dakin HD. Über die Arginase. Z Physiol Chem. 1904;41:321–331. 2. Krebs HA, Johnson WA. Metabolism of ketonic acids in animal tissues. Biochem J. 1937;31:645–660. 3. Krebs HA, Johnson WA. The role of citric acid in intermediate metabolism in animal tissues. FEBS Lett. 1980;117 Suppl:K1–K10. 4. Annau E, Banga I, Gozsy B, et al. Über die Bedeutung der Fumarsaure fur die tierische Gewabsatmung. Z Physiol Chem. 1935;235:1–68. 5. Martius C, Knoop F. Der physiologische Abbau der Citronensaure. Vorläufige mitteilung. Z Physiol Chem. 1937;246:1–11. 6. Martius C. Über den Abbau der Citronensaure. Z Physiol Chem. 1937;247:104–110. 7. Wagner-Jauregg T, Rauen H. Die Dehydrierung der Citronensäure und der Iso-Citronensäure durch Gerkensamen-Dehydrase. Z Physiol Chem. 1935;237:227. 8. Kornberg HL, Krebs HA. Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature. 1957;179:988–991. 9. Kornberg H. Krebs and his trinity of cycles. Nat Rev Mol Cell Biol. 2000;1:225–228. 10.Blaschko H. Obituary Hans Adolf Krebs (1900–1981). Rev Physiol Biochem Pharmacol. 1983;98:1–9. 11.Krebs HA, Martin A. Hans Krebs, Reminiscences and Reflections. Oxford: Oxford University Press; 1981. 12.Munnich A. Casting an eye on the Krebs cycle. Nat Genet. 2008;40:1148–1149. 13.Hartong DT, Dange M, McGee TL, et al. Insights from retinitis pigmentosa into the roles of isocitrate dehydrogenases in the Krebs cycle. Nat Genet. 2008;40:1230–1234. 14.Haworth JC, Perry TL, Blass JP, et al. Lactic acidosis in three sibs due to defects in both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. Pediatrics. 1976;58:564–572. June 2010 ■ Volume 41 Number 6 ■ LABMEDICINE 379 Luminaries 15.Elpeleg O, Miller C, Hershkovitz E, et al. Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion. Am J Hum Genet. 2005;76:1081–1086. 16.Ostergaard E, Christensen E, Kristensen E, et al. Deficiency of the alpha subunit of succinate-coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion. Am J Hum Genet. 2007;81:383–387. 17.Bourgeron T, Rustin P, Chretien D, et al. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat Genet. 1995;11:144–149. 18.Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science. 2000;287:848–851. 20.Niemann S, Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet. 2000;26:268–270. 21.Bourgeron T, Chretien D, Poggi-Bach J, et al. Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency. J Clin Invest. 1994;93:2514–2518. 22.Krebs HA, Johnson WA. The role of citric acid in intermediate metabolism in animal tissues. Enzymologia. 1937;4:148–156. 23.Green DE. The malic dehydrogenase of animal tissues. Biochem J. 1936;30:2095–2110. 19.Astuti D, Latif F, Dallol A, et al. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet. 2001;69:49–54. Note from the Editor-in-Chief Those of us who took a Biochemistry course remember fondly, and in some cases, perhaps not so fondly, the requirement to be able to reproduce all of the biochemical conversions in the Krebs cycle, including the structure of each intermediate! In the 1970s, the premier textbook of biochemistry was Biochemistry: The Molecular Basis of Cell Structure and Function by Albert L. Lehninger of The Johns Hopkins University School of Medicine, in which the “Krebs Cycle” was a prominent component. Moreover, the Warburg apparatus, invented by Krebs’ mentor, Dr. Otto Warburg, was not an uncommon sight in many university biochemistry laboratories. In the Spring of 1981, I had the distinct privilege of meeting Dr. Krebs during his visit to Harvard Medical School as the invited lecturer for the 1980-81 Dunham lecture series (see photos; Hans Krebs is, of course, the one on the right in this photo). Unfortunately, Dr. Krebs died the following November of natural causes; however, his legacy lives on, as no serious student of biochemistry can, or should, avoid a detailed study of the Krebs cycle. In the same year as his death, his book,* in collaboration with Roswitha Schmid, honoring the memory and influence of his mentor, Otto Warburg, was published. Photo of Sir Hans A. Krebs (right) and Dr Frank H. Wians, Jr (left), May 1981. *Krebs H. Otto Warburg: Cell Physiologist, Biochemist, and Eccentric. Oxford: Clarendon Press; 1981. Program from Krebs’ lecture at Harvard Medical School, May 1981. 380 LABMEDICINE ■ Volume 41 Number 6 ■ June 2010 labmedicine.com