Download Luminaries - Oxford Academic

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