Introduction Hypothesis Evidence Questions Refs

Unit 2: Cell and Molecular Biology

Lecture 2.xx: Krebs Cycle

John D. Nagy

BIO 181: General Biology for Majors, Scottsdale Community College

2019 Revision

John Nagy Lec 2.xx: Krebs Cycle 1/27

Introduction Hypothesis Evidence Questions Refs

Outline

1 IntroductionBeginningsProblem

2 HypothesisEarly cluesThe hypothesis of Hans Adolf Krebs and colleagues

3 EvidencePyruvateMalonate and Fumarate

4 Questions and AnswersPYR oxidationLocationSummary

5 References and Image SourcesImagesLiterature cited

John Nagy Lec 2.xx: Krebs Cycle 2/27

Introduction Hypothesis Evidence Questions Refs Beginnings 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 [3])Earliest expression of our modernunderstanding of the heart’s rolein blood circulation.

John Nagy Lec 2.xx: Krebs Cycle 3/27

Introduction Hypothesis Evidence Questions Refs Beginnings Problem

Joseph Priestley’s experiment

In 1770s, Joseph Priestley [12] discovers that, in a sealed chamber,

candle flames and a mouse both die at the same instant; but

introduce a growing plant, and both survive indefinitely.

Therefore, a gas is depleted by flames and living animals, but isreplenished by plants.

What is the gas?

John Nagy Lec 2.xx: Krebs Cycle 4/27

Introduction Hypothesis Evidence Questions Refs Beginnings 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 2.xx: Krebs Cycle 5/27

Introduction Hypothesis Evidence Questions Refs Beginnings Problem

True or false?

Oxygen is needed by glycolysis, and carbon dioxide is producedby glycolysis?

What metabolic process is oxygen dependent but produces CO2?

John Nagy Lec 2.xx: Krebs Cycle 6/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 7/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 8/27

Introduction Hypothesis Evidence Questions Refs 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 [14]) to be related asfollows:

What do you predict would happen if we added oxaloacetate toliving mammalian cells?

John Nagy Lec 2.xx: Krebs Cycle 9/27

Introduction Hypothesis Evidence Questions Refs Early clues Krebs

A key result in 1937

In 1937, Martius and Knoop published 2 papers [10, 11]announcing the discovery that tricaboxylic acids (like citric acidfrom fruits) were connected to succinic acid via the followingreactions:

Note: This is the modern understanding. Martius and Knoop’sversion was less detailed.

John Nagy Lec 2.xx: Krebs Cycle 10/27

Introduction Hypothesis Evidence Questions Refs Early clues Krebs

Summary of knowledge in 1937

John Nagy Lec 2.xx: Krebs Cycle 11/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 12/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 13/27

Introduction Hypothesis Evidence Questions Refs Early clues Krebs

Krebs and Johnson’s hypothesis [7]

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 2.xx: Krebs Cycle 14/27

Introduction Hypothesis Evidence Questions Refs Pyruvate Malonate and Fumarate

Prediction: if true, pyruvate will be oxidized

Theoretical yield from complete oxidation of pyruvic acid

C3H4O3 + 2.5O2 −→ 3CO2 + 2H2O

Krebs and Eggleston (1940) [6]:

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.

Note: one “turn” of the cycle produces how many CO2?

Does this observation support, contradict or say nothing aboutKrebs and Johnson’s [7] hypothesis?

John Nagy Lec 2.xx: Krebs Cycle 15/27

Introduction Hypothesis Evidence Questions Refs Pyruvate Malonate and Fumarate

Evidence from a metabolic poison

CO2

Malonate (Malonate (not malatenot malate) inhibits) inhibitssuccinate dehydrogenasesuccinate dehydrogenase

XXMalonate inhibits succinate dehydrogenase.

Malonate inhibits oxidative metabolism [5].

Does this observation support, contradict or say nothingabout Krebs and Johnson’s hypothesis?

John Nagy Lec 2.xx: Krebs Cycle 16/27

Introduction Hypothesis Evidence Questions Refs Pyruvate Malonate and Fumarate

Experiment based on malonate

CO2

Malonate (Malonate (not malatenot malate) inhibits) inhibitssuccinate dehydrogenasesuccinate dehydrogenase

XX

Experiment

Predict how PYR consumption would change if cells are given

excess fumarate;

malonate;

both fumarate and malonate at the same time.

John Nagy Lec 2.xx: Krebs Cycle 17/27

Introduction Hypothesis Evidence Questions Refs Pyruvate Malonate and Fumarate

Results of the malonate experiment

More from Krebs and Eggleston (1940) [6]:

“Cont” = Control (nothingadded)

“F” = FUM only added

“P” = 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 2.xx: Krebs Cycle 18/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 19/27

Introduction Hypothesis Evidence Questions Refs PYR oxidation Location Summary

Crucial role of coenzyme A in PYR oxidation

Fritz Lipmann and colleagues discover coenzyme A [9] andelucidates its role in oxidation of PYR [8, 13].

Severo Ochoa and Feodor Lynen “established acetylcoenzyme A as the intermediate which reacts withoxaloacetate to form citrate” (Krebs 1970 [5]).

John Nagy Lec 2.xx: Krebs Cycle 20/27

Introduction Hypothesis Evidence Questions Refs 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 [1, 4].)

John Nagy Lec 2.xx: Krebs Cycle 21/27

Introduction Hypothesis Evidence Questions Refs PYR oxidation Location Summary

Summary of current theory

John Nagy Lec 2.xx: Krebs Cycle 22/27

Introduction Hypothesis Evidence Questions Refs 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 2.xx: Krebs Cycle 23/27

Introduction Hypothesis Evidence Questions Refs Images Literature cited

Image sources I

Slide 1: SciencePhotoLibrary (www.sciencephoto.com/media/507286/view)

Slide 3:

Left: Portrait of William Harvey. Engraving by Edward Scriven from a paintingby Cornelius Johnson.Image from Harvey (1628) [3]

Slide 4:

(Left): Portrait of Joseph Priestley. Engraving by William Holl the Elder from apainting by Gilbert Stuart.(Right): Aleesa Monaco.

Slide 5: Figure 42.1, page 875 of Freeman et al. (2017) [2].

Slide 6: www.onlinebiologynotes.com/glycolysis-steps-diagram-and-enzymes-involved.

Slides 7, 8, 18: J. D. Nagy.

Slides 9-11, 13, 14, 16, 17, 19: Adapted frommicrobiologyinfo.com/krebs-citric-acid-cycle-steps-by-steps-explanation.

Slide 12: National Portrait Gallery(www.npg.org.uk/collections/search/portrait/mw14125/Sir-Hans-Adolf-Krebs).

Slide 20: Figure 9.9, page 197 of Freeman et al. (2017) [2].

Slide 22: Figure 9.10 of Freeman et al. (2017) [2].

Slide 23: NobelPrize.org (www.nobelprize.org/prizes/medicine/1953/summary).

John Nagy Lec 2.xx: Krebs Cycle 24/27

Introduction Hypothesis Evidence Questions Refs Images Literature cited

References I

Daniel K. Bricker, Eric B. Taylor, John C. Schnell, Thomas Orsak, Audery Boutron,

Yu-Chan Chen, James E. Cox, Caleb M. Cardon, Jonathan G. Van Vranken, NoalDephoure, Claire Redin, Sihem Boudina, Steven P. Gygi, Miche Brivet, Carl S.Thummel, and Jared Rutter.A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, andhumans.Science, 337:96–100, 2012.

Scott Freeman, Kim Quillin, Lizabeth Allison, Michael Black, Greg Podgorski, Emily

Taylor, and Jeff Carmichael.Biological Science.Benjamin Cummings, San Francisco, 6th edition, 2017.

William Harvey.

Exercitatio Anaatomica: De Motu Cordis et Sanguinis in Animalibus, 1628.Charles C. Thomas, Springfield, IL, 1928.

Sebastien Herzig, Etienne Raemy, Sylvie Montessuit, Jean-Luc Veurthey, Nicola

Zamboni, Benedikt Westermann, Edmund R. S. Kunji, and Jean-Claude Martinou.Identification and functional expression of the mitochondrial pyruvate carrier.Science, 337:93–97, 2012.

Hans Adolf Krebs.

The history of the Tricarboxylic Acid Cycle.Perspect. Biol. Med., 14(1):154–172, 1970.

Hans Adolf Krebs and Leonard V. Eggleston.

The oxidation of pyruvate in pigeon breast muscle.Biochem. J., 34(3):442–459, 1940.

John Nagy Lec 2.xx: Krebs Cycle 25/27

Introduction Hypothesis Evidence Questions Refs Images Literature cited

References II

Hans Adolf Krebs and William A. Johnson.

The role of citric acid in intermdiate metabolism in animal tissues.Enzymologia, 4:148–156, 1937.

Fritz Lipmann.

On chemistry and function of coenzyme A.Bacteriol. Rev., 17(1):1–16, 1953.

Fritz Lipmann, Nathan O. Kaplan, G. David Novelli, L. Constance Tuttle, and

Beverly M. Guirard.Isolation of coenzyme A.J. Biol. Chem., 186(1):235–243, 1950.

C. Martius.

The metabolism of citric acid.Z. Physiol. Chem, 247:104–110, 1937.

C. Martius and F. Knoop.

Physiological breakdown of citric acid.Z. Physiol. Chem., 246:1–11, 1937.

Joseph Priestley.

Experiments and Observations on Different Kinds of Air.J. Johnson, London, 1774-1786.

E. R. Stadtman, G. David Novelli, and Fritz Lipmann.

Coenzyme A function in and acetyl transfer by the phosphotransacetylase system.J. Biol. Chem., 191(1):365–376, 1951.

John Nagy Lec 2.xx: Krebs Cycle 26/27

Introduction Hypothesis Evidence Questions Refs Images Literature cited

References III

Torsten Thunberg.

Intermediary metabolism and the enzymes concerned therein.Skand. Arch. Physiol., 40:1–91, 1920.

John Nagy Lec 2.xx: Krebs Cycle 27/27