Transcript Document

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)
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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?
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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
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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!