Biochemistry - I · 2019-06-28 · 616 The Citric Acid Cycle 16.1 Production of Acetyl-CoA...

Based on Profs. Kevin Gardner & Reza Khayat 1

Biochemistry - I

Mondays and Wednesdays 9:30-10:45 AM (MR-1307)

SPRING 2017

Lectures 19-20

Outline

• Pyruvate is oxidized to Acetyl-CoA and CO2 • The pyruvate dehydrogenase complex requires five coenzymes • The pyruvate dehydrogenase complex consists of 3 distinct enzymes • The complex uses substrate channeling to maintain substrates on its surface • The Citric Acid Cycle • Anaerobic and Aerobic TCA • Stoichiometry of glucose oxidation • The amphibolic TCA • Replenishing TCA • Biological tethers • Regulation of TCA

Why learn about the Citric Acid Cycle? The citric acid cycle is considered to be the “hub” of metabolism as it is generates energy (catabolism) and key intermediates for synthesis (anabolism).

3Chapter 16 - The Citric Acid Cycle

• One pathway, many names: citric acid cycle = tricarboxylic acid (TCA) cycle = the Krebs cycle

• Cellular respiration: the consumption of nutrients by cells to produce CO2 and H2O (occurs in 3 steps)

• acetyl-CoA production • acetyl-CoA oxidation • electron transfer and oxidative phosphorylation

• ATP production occurs throughout, but greatest production occurs in last step (2.5 molecules of ATP per pair of e-)

• Amino acids, fatty acids, sugars are degraded to acetate and fed into TCA. Also possible to directly enter cycle as a TCA intermediate

Catabolism of Amino Acids, Fats, and Carbohydrates


• The reaction is an oxidative decarboxylation – irreversible in cell

• Pyruvate is shuttled from cytosol into mitochondria of eukaryotic cell

• Pyruvate dehydrogenase complex (Ac-CoA) • Located in mitochondria of eukaryotic

cell • Cluster of three enzymes • Five cofactors derived from vitamins • Regulated by allosteric and covalent

modification

Stage 1: Pyruvate is Oxidized to Acetyl-CoA and CO2


• Five coenzymes or prosthetic groups: thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD), and lipoate.

• The coenzymes function to activate the substrate, analogously to how UDP activates glucose and ribose for performing chemistry

Stage 1: Pyruvate is Oxidized to Acetyl-CoA and CO2

Chapter 16 - The Citric Acid Cycle

7

Coenzyme A activates the acetyl-CoA for subsequent reactions in TCA cycle (B5)

Lipoic acid (derived from octanoic acid) possesses two sulfurs that are oxidized and reduced to carry a hydrogen and an acetyl group. Covalently modifies Lys of E2

NAD+ is an oxidizing agent that carries a proton and two electrons to the e- transport chain for ATP synthesis (B3)

FAD+2 is also an oxidizing agent that carries 2e- and 2H+. These are passed to NAD+ (B2)

TPP accepts the decarboxylated form of pyruvate (B1)

F

A

D

Four come from B

vitamins Chapter 16 - The Citric Acid Cycle

Pyruvate Dehydrogenase Complex

8

• A complex of three proteins • E1 (yellow) pyruvate dehydrogenase • E2 (green) dihydrolipoly transacetylase • E3 (red) dihydrolipoly dehydrogenase

• 4.5 MDa MW, 45 nm diameter • Lipoate is attached to lysine side chain of E2 • Two regulatory proteins also in complex: a protein kinase and

phosphoprotein phosphatase


Step 1: C1 Released as CO2, C2 attached to TPP


Step 2: Oxidation at C2

10

• Step 2: the C2 carbon is oxidized from an alcohol to a ketone. The two electrons removed reduce the -S-S- of a lipoyl group to two thiol groups (-SH). The acetate group is first esterified to one of the lipoly –SH groups (= thioester) then transesterified to CoA to form acetyl-CoA in step 3.

H+ picked up by E2


Step 3: Complete Formation of Acetyl-CoA

11

• Step 3: Energy of oxidation drives the formation of a high-energy thioester of acetate = acetyl CoA


Steps 4 & 5: Regenerate oxidized lipoid groups

12

• Steps 4 and 5: Electron transfers regenerate the oxidized (disulfide) form of the lipoyl groups on lipoid acid for another round of oxidation. The electrons from pyruvate pass through FAD to NADH.

• Substrate shuttling: Swinging lipoly arms attach intermediates to enzyme, keeping intermediates within the complex


Diseases Affecting Stage 1

13

Thiamine deficiency is a disease that can arise from poor diet, leading to the inability to oxidize pyruvate normally. This slows down the TCA and requires other pathways to get into TCA. • Beriberi: nutritional deficiencies of TPP (vitamin B1):

• Thiamine is not present in fats or highly refined sugars and is present sparingly in cassava. Foods containing thiaminases, such as milled rice, shrimp, mussels, clams, fresh fish, and raw animal tissues, decrease absorption.

• Good sources: Whole-grain foods / Meat/fish/poultry/eggs / Milk and milk products / Vegetables (ie, green, leafy vegetables; beets; potatoes) / Legumes (ie, lentils, soybeans, nuts, seeds) / Orange and tomato juices

• Alcoholism: Inhibits B1 uptake in intestine, storage in liver


• One pathway, many names: citric acid cycle = tricarboxylic acid (TCA) cycle = the Krebs cycle

• Cellular respiration: the consumption of nutrients by cells to produce CO2 and H2O (occurs in 3 steps)

• acetyl-CoA production • acetyl-CoA oxidation • electron transfer and oxidative phosphorylation

• ATP production occurs throughout, but greatest production occurs in last step (2.5 molecules of ATP per pair of e-)

• Amino acids, fatty acids, sugars are degraded to acetate and fed into TCA. Also possible to directly enter cycle as a TCA intermediate

Catabolism of Amino Acids, Fats, and Carbohydrates


The Citric Acid Cycle

15

1.1 Cellular Foundations 5

evolved from the same branch that gave rise to the Ar-chaea; eukaryotes are therefore more closely related toarchaea than to bacteria.

Within the domains of Archaea and Bacteria aresubgroups distinguished by their habitats. In aerobichabitats with a plentiful supply of oxygen, some residentorganisms derive energy from the transfer of electronsfrom fuel molecules to oxygen. Other environments areanaerobic, virtually devoid of oxygen, and microorgan-isms adapted to these environments obtain energy bytransferring electrons to nitrate (forming N2), sulfate(forming H2S), or CO2 (forming CH4). Many organismsthat have evolved in anaerobic environments are obli-gate anaerobes: they die when exposed to oxygen. Oth-ers are facultative anaerobes, able to live with orwithout oxygen.

We can classify organisms according to how they ob-tain the energy and carbon they need for synthesizingcellular material (as summarized in Fig. 1–5). There aretwo broad categories based on energy sources: pho-totrophs (Greek trophe, “nourishment”) trap and usesunlight, and chemotrophs derive their energy fromoxidation of a chemical fuel. Some chemotrophs, thelithotrophs, oxidize inorganic fuels—HS! to S0 (ele-mental sulfur), S0 to SO4

!, NO2! to NO3

!, or Fe2" to Fe3",for example. Organotrophs oxidize a wide array of or-ganic compounds available in their surroundings. Pho-totrophs and chemotrophs may also be divided intothose that can obtain all needed carbon from CO2 (au-totrophs) and those that require organic nutrients(heterotrophs).

Escherichia coli Is the Most-Studied Bacterium

Bacterial cells share certain common structural features,but also show group-specific specializations (Fig. 1–6).E. coli is a usually harmless inhabitant of the humanintestinal tract. The E. coli cell is about 2 !m long anda little less than 1 !m in diameter. It has a protectiveouter membrane and an inner plasma membrane thatencloses the cytoplasm and the nucleoid. Between theinner and outer membranes is a thin but strong layer ofa polymer (peptidoglycan) that gives the cell its shapeand rigidity. The plasma membrane and the layers out-side it constitute the cell envelope. We should notehere that in archaea, rigidity is conferred by a differenttype of polymer (pseudopeptidoglycan). The plasmamembranes of bacteria consist of a thin bilayer of lipid

molecules penetrated by proteins. Archaeal membraneshave a similar architecture, but the lipids are strikinglydifferent from those of bacteria (see Fig. 10–12).

Ribosomes Bacterial ribosomes are smaller thaneukaryotic ribosomes, but serve the same function—protein synthesis from an RNA message.

Nucleoid Contains a single,simple, long circular DNAmolecule.

Pili Providepoints ofadhesion tosurface ofother cells.

FlagellaPropel cellthrough itssurroundings.

Cell envelopeStructure varieswith type ofbacteria.

Gram-negative bacteriaOuter membrane;peptidoglycan layer

Outer membrane

Peptidoglycan layerInner membraneInner membraneInner membrane

Gram-positive bacteriaNo outer membrane;thicker peptidoglycan layer

CyanobacteriaGram-negative; tougherpeptidoglycan layer;extensive internalmembrane system withphotosynthetic pigments

ArchaeaNo outer membrane;pseudopeptidoglycan layeroutside plasma membrane

Peptidoglycan layerInner membrane

FIGURE 1– 6 Common structural features of bacterial cells. Because ofdifferences in cell envelope structure, some bacteria (gram-positivebacteria) retain Gram’s stain (introduced by Hans Christian Gram in1882), and others (gram-negative bacteria) do not. E. coli is gram-negative. Cyanobacteria are distinguished by their extensive internalmembrane system, which is the site of photosynthetic pigments. Al-though the cell envelopes of archaea and gram-positive bacteria looksimilar under the electron microscope, the structures of the membranelipids and the polysaccharides are distinctly different (see Fig. 10–12).

The Foundations of Biochemistry6

The cytoplasm of E. coli contains about 15,000 ri-bosomes, various numbers (10 to thousands) of copiesof each of 1,000 or so different enzymes, perhaps 1,000organic compounds of molecular weight less than 1,000

(metabolites and cofactors), and a variety of inorganicions. The nucleoid contains a single, circular molecule ofDNA, and the cytoplasm (like that of most bacteria)contains one or more smaller, circular segments of DNA

Ribosomes are protein-synthesizing machines

Peroxisome oxidizes fatty acids

Lysosome degrades intracellulardebris

Transport vesicle shuttles lipidsand proteins between ER, Golgi,and plasma membrane

Golgi complex processes,packages, and targets proteins toother organelles or for export

Smooth endoplasmic reticulum(SER) is site of lipid synthesisand drug metabolism

Nucleus contains thegenes (chromatin)

RibosomesNuclearenvelope Cytoskeleton

Cytoskeleton supports cell, aidsin movement of organelles

Golgicomplex

Nucleolus is site of ribosomalRNA synthesis

Rough endoplasmic reticulum(RER) is site of much proteinsynthesis

Mitochondrion oxidizes fuels toproduce ATP

Plasma membrane separates cellfrom environment, regulatesmovement of materials into andout of cell

Chloroplast harvests sunlight,produces ATP and carbohydrates

Starch granule temporarily storescarbohydrate products ofphotosynthesis

Thylakoids are site of light-driven ATP synthesis

Cell wall provides shape andrigidity; protects cell fromosmotic swelling

Cell wall of adjacent cellPlasmodesma provides pathbetween two plant cells

Nuclear envelope segregateschromatin (DNA ! protein)from cytoplasm

Vacuole degrades and recyclesmacromolecules, storesmetabolites

(a) Animal cell

(b) Plant cell

Glyoxysome contains enzymes ofthe glyoxylate cycle

FIGURE 1– 7 Eukaryotic cell structure. Schematic illustrations of twomajor types of eukaryotic cell: (a) a representative animal cell and(b) a representative plant cell. Plant cells are usually 10 to 100 !m indiameter—larger than animal cells, which typically range from 5 to 30 !m.

Structures labeled in red are unique to either animal or plant cells.Eukaryotic microorganisms (such as protists and fungi) have structuressimilar to those in plant and animal cells, but many also contain spe-cialized organelles not illustrated here.

The Citric Acid Cycle616

16.1 Production of Acetyl-CoA (Activated Acetate)In aerobic organisms, glucose and other sugars, fattyacids, and most amino acids are ultimately oxidizedto CO2 and H2O via the citric acid cycle and the

respiratory chain. Before entering the citric acid cy-cle, the carbon skeletons of sugars and fatty acidsare degraded to the acetyl group of acetyl-CoA, theform in which the cycle accepts most of its fuel in-put. Many amino acid carbons also enter the cyclethis way, although several amino acids are degradedto other cycle intermediates. Here we focus on howpyruvate, derived from glucose and other sugars byglycolysis, is oxidized to acetyl-CoA and CO2 by thepyruvate dehydrogenase (PDH) complex, a clus-ter of enzymes—multiple copies of each of three en-zymes—located in the mitochondria of eukaryoticcells and in the cytosol of bacteria.

A careful examination of this enzyme complex is re-warding in several respects. The PDH complex is a clas-sic, much-studied example of a multienzyme complex inwhich a series of chemical intermediates remain boundto the enzyme molecules as a substrate is transformedinto the final product. Five cofactors, four derived fromvitamins, participate in the reaction mechanism. Theregulation of this enzyme complex also illustrates how a combination of covalent modification and allostericreg-ulation results in precisely regulated flux through a metabolic step. Finally, the PDH complex is the proto-type for two other important enzyme complexes: !-ketoglutarate dehydrogenase, of the citric acid cycle,and the branched-chain !-keto acid dehydrogenase,of the oxidative pathways of several amino acids (seeFig. 18–28). The remarkable similarity in the proteinstructure, cofactor requirements, and reaction mecha-nisms of these three complexes doubtless reflects acommon evolutionary origin.

Pyruvate Is Oxidized to Acetyl-CoA and CO2

The overall reaction catalyzed by the pyruvate dehydro-genase complex is an oxidative decarboxylation, anirreversible oxidation process in which the carboxylgroup is removed from pyruvate as a molecule of CO2

and the two remaining carbons become the acetyl groupof acetyl-CoA (Fig. 16–2). The NADH formed in this re-action gives up a hydride ion (:H!) to the respiratorychain (Fig. 16–1), which carries the two electrons to

"CoA-SH

NADH

Acetyl-CoA

M DO

AC

S-CoANAD"

AC

Pyruvate

CH3

M

P

O

AO

C

!O

CH3

CO2

D

#G$% & !33.4 kJ/mol

pyruvate dehydrogenasecomplex (E1 " E2 " E3)

TPP,lipoate,

FAD

FIGURE 16–2 Overall reaction catalyzed by the pyruvate dehydrogenasecomplex. The five coenzymes participating in this reaction, and the threeenzymes that make up the enzyme complex, are discussed in the text.

NADH,FADH2

(reduced e! carriers)

Respiratory(electron-transfer)

chain

ATPADP + Pi

H2O

Stage 3Electron transfer

and oxidativephosphorylation

Citricacid cycle

Stage 2Acetyl-CoAoxidation

Acetyl-CoA

Oxaloacetate

CO2

pyruvatedehydrogenasecomplex

Pyruvate

Glycolysis

Fattyacids

Aminoacids

e!

Stage 1Acetyl-CoAproduction

2H+ +

e!

e!

e!

CO2

CO2

e!

e!

e!

e!

e!

12O2

Glucose

Citrate

FIGURE 16–1 Catabolism of proteins, fats, and carbohydrates in thethree stages of cellular respiration. Stage 1: oxidation of fatty acids,glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation ofacetyl groups in the citric acid cycle includes four steps in which elec-trons are abstracted. Stage 3: electrons carried by NADH and FADH2

are funneled into a chain of mitochondrial (or, in bacteria, plasmamembrane–bound) electron carriers—the respiratory chain—ultimatelyreducing O2 to H2O. This electron flow drives the production of ATP.


Stage 2: The TCA cycle

16

• The recycling center of the cell • Takes in material, generates energy & material for synthesis & waste • The process is amphibolic: both catabolic and anabolic • Two carbons enter (acetyl CoA) and two leave (CO2) • In the first step, acetyl CoA is fused to oxaloacetate. OAA is present in

cells in very low concentration, allowing control of TCA cycle by regulating formation of newly synthesized oxaloacetate molecules

• Formation of acetyl CoA by the pyruvate dehydrogenase complex (PDC) is also under regulatory control: inactivated by a kinase that is under allosteric control by ATP (activated), and activated by a phosphorylase

• Process is highly oxidizing: four of the eight steps are oxidation reactions and are used to reduce FAD+ and NAD+

• Four and five-carbon intermediates are precursors for a variety of products


The Citric Acid Cycle


NAD Oxidation and Reduction

21

NADP has PO4 here (anabolic reactions)

Water soluble

+ H+

FYIChapter 16 - The Citric Acid Cycle

+ +

22

Water insoluble


FAD Oxidation and Reduction

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No substrate/cofactor Exergonic (thioester cleavage) Highly regulated ∆G=-32.2kJ/mol

No substrate/cofactor Near equilibrium Not regulated ∆G=13.3kJ/mol

Yes, NAD+ and CoA Exergonic Highly regulated ∆G=-33.5kJ/mol (both reactions 3&4) KDH is similar to PDH

Yes, GDP and Pi Near equilibrium Not regulated ∆G=-2.9kJ/mol

Yes, FAD Near equilibrium Not regulated ∆G=0kJ/mol

No substrate/cofactor Near equilibrium Not regulated ∆G=-3.8kJ/mol

Yes, NAD+ Endergonic Regulated ∆G=29.7kJ/mol

Yes, NAD+ and Mn2+ Exergonic Highly regulated

Aerobic TCA


2 CO2

Stoichiometry of Glucose Oxidation


Anaerobic TCA


Amphibolic TCA

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Catabolic Anabolic


Replenishing TCA

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• TCA-replenishing (anaplerotic) reactions make intermediates for TCA • enzymes are highly regulated • pyruvate carboxylase is activated by acetyl-CoA (positive allosteric modulator).

Excess acetyl-CoA means TCA is backed up. Thus More oxaloacetate is created to initiate more cycles

• PEP carboxylase is activated by F1,6BP (an intermediate in glycolysis). Accumulation of F1,6BP signals that more TCA cycles need to be initiated for oxidation of glucose to CO2 (i.e. TCA is limiting glycolysis)


Biological Tethers

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• Cofactors that are covalently attached to the enzyme

• Usually contain long linkers so that the cofactor can swing from one active site to another active site

• Lipoyllysine swings back and forth between E1 and E3


Regulation of TCA

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• Regulation ensures production of intermediates at rates which keep cell in a stable steady state (no backups) while avoiding wasteful overproduction

• Regulation of the pyruvate dehydrogenase complex occurs prior to TCA

• Regulation occurs with the most exergonic reactions

• Rate of flux governed by: • Substrate availability • Accumulating products • Allosteric feed back inhibition

• Ca+2, muscle contraction


Citrate Regulates TCA and R3 of Glycolysis

ATP substrate

allosteric regulator

PFK-1 is the “committed” step to glycolysis and thus under allosteric regulation (graph on the right) Has a catalytic and allosteric site (shown bottom right is ADP in blue and fructose 1,6-bisphosphate in yellow) Inhibited allosterically by ATP (at levels that signal enough is being produced) High [ATP] lowers affinity of enzyme for its substrate (right)

31Chapter 15 - Metabolic Regulation Chapter 16 - The Citric Acid Cycle

Biochemistry - I · 2019-06-28 · 616 The Citric Acid Cycle 16.1 Production of Acetyl-CoA...

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