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Introduction Hypothesis Evidence Questions Lecture 4.07: Krebs Cycle John D. Nagy Scottsdale Community College BIO 181, General Biology for Majors John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions
Lecture 4.07: Krebs Cycle
John D. Nagy
Scottsdale Community College
BIO 181, General Biology for Majors
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions
Outline
1 IntroductionBeginningsSynthesisProblem
2 HypothesisEarly cluesThe hypothesis of Hans Adolf Krebs and colleagues
3 EvidenceMalonatePyruvateFumarate
4 Questions and AnswersPYR oxidationLocationSummary
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Beginnings Synthesis Problem
William Harvey’s discovery
Exercitatio Anatomica de MotuCordis et Sanguinis in Animalibus(“Anatomical Exercises: The Mo-tion of the Heart and Blood in An-imals;” 1628)Earliest expression of our modernunderstanding of the heart’s rolein blood circulation.
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Beginnings Synthesis Problem
Joseph Priestley’s experiment
In 1770s, Joseph Priestley discoversthat plants produce a gas consumedby fire but required by mice to sur-vive.What is the gas?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Beginnings Synthesis Problem
Modern synthesis of Harvey and Priestley
Respiratory gas exchange:
O2 is carried by blood from external environment to tissues.
CO2 is carried by blood from tissues to external environment.
Why?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Beginnings Synthesis Problem
True or false?
Oxygen is needed by glycolysis, and carbon dioxide is producedby glycolysis?
What metabolic process is oxygen dependent?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Early evidence pointing in the right direction
Pyruvate, acetate, succinate, fumarate and malate are all organicacids (actually, conjugate bases of organic acids). This is whathappens when they are added to living mammalian cells.
What does this evidence teach us?John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
What we learn from this evidence
Discovery (1920s and 1930s)
When certain organic acids are added to living mammalian(and avian) cells, O2 is consumed and CO2 is produced.
⇒ Respiration has something to do with organic acidmetabolism. But what?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Evidence from organic chemistry
Intriguingly, when these discoveries were made (in the 1930s),pyruvate was known to be a product of glycolysis, and theothers were suspected (by Thunberg) to be related as follows:
What do you predict would happen if we added oxaloacetate toliving mammalian cells?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
A key result in 1937
In 1937, Martius and Knoop published 2 papers announcing thediscovery that tricaboxylic acids (like citric acid from fruits)were connected to succinic acid via the following reactions:
Note: This is the modern understanding. Martius and Knoop’sversion was less detailed.
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Summary of knowledge in 1937
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Hans Adolf Krebs and manometry
H.A. Krebs (student of Otto Warburg) and his manometer—away to precisely measure gases consumed and produced byliving cells.
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Observation and question
Krebs and Johnson (1937) showed that citrate isKrebs and Johnson (1937) showed that citrate ismade if oxaloacetate is fed to the cells. made if oxaloacetate is fed to the cells. Where do the 2 extra carbons come from?Where do the 2 extra carbons come from?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Early clues Krebs
Krebs and Johnson’s hypothesis
Krebs and Johnson hypothesized that pyruvate fromKrebs and Johnson hypothesized that pyruvate fromglycolysis supplies the needed C. This would make aglycolysis supplies the needed C. This would make a metabolic metabolic cyclecycle connecting anaerobic with aerobic connecting anaerobic with aerobicmetabolism.metabolism.
CO2
Now, they need to assess evidence for this hypothesis.
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Malonate Pyruvate Fumarate
Clues from a metabolic poison
Malonate (Malonate (not malatenot malate) inhibits) inhibitssuccinate dehydrogenasesuccinate dehydrogenase
XXHow would malonate affect living cells, do you predict?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Malonate Pyruvate Fumarate
Effects of malonate
Malonate (Malonate (not malatenot malate) inhibits) inhibitssuccinate dehydrogenasesuccinate dehydrogenase
XXAdd malonate to living cells
Cells cease consuming oxygen.
Cells cease oxidizing pyruvate.
Does this observation support, contradict or say nothing aboutKrebs and Johnson’s hypothesis?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Malonate Pyruvate Fumarate
Feeding cells pyruvate
Complete oxidation of pyruvic acid
C3H4O3 + 2.5O2 −→ 3CO2 + 2H2O
Krebs and Eggleston (1940):
Fed cells pyruvate and measured O2 consumption and CO2
release.
Expected results (see equation above):2.5 mol O2 consumed for every mol of pyruvate added.3 mol of CO2 released for every mol of pyruvate added.
Observed results:2.54 mol O2 consumed for every mol of pyruvate added.3.01 mol of CO2 released for every mol of pyruvate added.
⇒ Essentially all added pyruvate is oxidized. Pyruvate islike candy to a cell.
Does this observation support, contradict or say nothing aboutKrebs and Johnson’s hypothesis?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions Malonate Pyruvate Fumarate
The effect of fumarate
More from Krebs and Eggleston (1940):
“Cont” = Control (nothingadded)
“F” = FUM only added
“M” = Poison only added
“P+F L” = Poison + FUMlow concentration
“P+F M” = Poison + FUMmid concentration
“P+F H” = Poison + FUMhigh concentration
⇒ Fumarate counteracts the effect of the malonate poison in adose-dependent way.Does this observation support, contradict or say nothing aboutKrebs and Johnson’s hypothesis?
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions PYR oxidation Location Summary
First open question
Open questionOpen question: : How is PYR fed into the cycle?How is PYR fed into the cycle?
CO2
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions PYR oxidation Location Summary
Crucial role of coenzyme A in PYR oxidation
Fritz Lipmann discovers coenzyme A and elucidates its rolein oxidation of PYR.
Severo Ochoa and Feodor Lynen “established acetylcoenzyme A as the intermediate which reacts with OXALto form CIT” (Krebs 1970).
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions PYR oxidation Location Summary
Where does all this occur?
Enzymes of PYR oxidation and Krebs cycle are in the innermitochondrial membrane; rxns occur in mitochondrial matrix.
Glycolysis occurs in the cytosol.
PYR diffuses through outer mitochondrial membrane.
PYR actively transported across inner membrane. (Transporterdiscovered in 2012.)
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions PYR oxidation Location Summary
Summary of current theory
John Nagy Lec 4.07: Krebs Cycle
Introduction Hypothesis Evidence Questions PYR oxidation Location Summary
Shared Nobel Prize, Physiology/Medicine, 1953
Krebs (left) awarded “for his discovery of the citric acidcycle.”
Lipmann (right) awarded “or his discovery of co-enzyme Aand its importance for intermediary metabolism.”
John Nagy Lec 4.07: Krebs Cycle
