Download The Citric Acid Cycle

The Citric Acid Cycle
(How cells breathe!)
Lectures 17-18
Chem 464
Abrol Section
1
GoFormative Questions – 2017-04-12
• We have glucose present and the hormone used
gives energy anaerobic oxidation of glucose.
• We talked about how anaerobic oxidation comes
into play during adrenaline. Does energy storage
have anything to do with it also?
• Is Mg2+ the only metal used in glycolysis in bacteria?
(“Comparison of ability of Mg and Mn to activate the
key enzymes of Glycolysis.”, Wimhurst and Manchester
(1972) FEBS Letters 27, 321.
Fight or Flight System
https://web.archive.org/web/20130808004906/http://learn.genetics.utah.edu:80/content/begi
n/cells/fight_flight/
• Sensory nerve cells pass the perception of a threat, or stress, from the environment to the
hypothalamus in the brain, where neurosecretory cells transmit a signal to the pituitary
gland inciting cells there to release a chemical messenger ACTH into the bloodstream.
• Simultaneously, the hypothalamus transmits a nerve signal down the spinal cord. Both the
chemical messenger and nerve impulse will travel to the adrenal gland.
• Sitting atop the kidneys, the adrenal glands receive nerve and chemical signals initiated by
cells in the hypothalamus. Nerve signals activate the release of epinephrine into the blood.
• When chemical messengers arrive via the bloodstream, they dock on to receptors and begin
a cell signaling cascade that results in the production of cortisol.
• Cortisol is released into the blood stream where it begins signaling cascades in several cell
types, resulting in an increase in blood pressure, increase in blood sugar levels, and
suppression of the immune system.
• Signaling molecules from several origins work to provide an energetic boost in a variety of
ways. When epinephrine binds to receptors on liver cells, it triggers a signaling cascade that
produces glucose from larger sugar molecules.
• Circulating cortisol sets fatty acids free to be transformed into energy. These molecules are
rapidly excreted into the bloodstream, supplying a boost of readily available energy for
muscles throughout the body, priming them for exertion.
Metal Ions in Biology
Metal Ion
Mg2+
Zn2+
Mn2+
Ca2+
Pb2+
Size (CSD data)
0.65Å
0.71Å
0.74Å
0.99Å
1.12Å
CHAPTER 16:
The Citric Acid Cycle
Key topics:
–
–
–
–
–
Cellular respiration
Conversion of pyruvate to activated acetate
Reactions of the citric acid cycle
Regulation of the citric acid cycle
Conversion of acetate to carbohydrate precursors in
the glyoxylate cycle
“The History of the Tricarboxylic Acid Cycle”, H.A. Krebs (1970);
Perspectives in Biology and Medicine, 14: 154-170.
Only a small amount of energy available
in glucose is captured in glycolysis
Glycolysis
∆G′° = –146 kJ/mol
2
GLUCOSE
Full oxidation (+ 6 O2)
∆G′° = –2,840 kJ/mol
6 CO2 + 6 H2O
Cellular Respiration
• Process in which cells consume O2 and produce CO2
• Provides more energy (ATP) from glucose than glycolysis
• Also captures energy stored in lipids and amino acids
• Evolutionary origin: developed about 2.5 billion years ago
• Used by animals, plants, and many microorganisms
• Occurs in three major stages:
- acetyl CoA production
- acetyl CoA oxidation
- electron transfer and oxidative phosphorylation
Respiration: Stage 1
Acetyl-CoA Production
Generates some:
ATP, NADH,
FADH2
Respiration: Stage 2
Acetyl-CoA oxidation
Generates more
NADH, FADH2,
and one GTP
Respiration: Stage 3
Oxidative Phosphorylation
Generates
a lot of ATP
In eukaryotes, citric acid cycle occurs in
mitochondria
• Glycolysis occurs in the
cytoplasm
• Citric acid cycle occurs in the
mitochondrial matrix†
• Oxidative phosphorylation
occurs in the inner membrane
†Except
succinate dehydrogenase, which
is located in the inner membrane
Conversion of Pyruvate to Acetyl-CoA
• Net Reaction:
– Oxidative decarboxylation of pyruvate
– First carbons of glucose to be fully oxidized
• Catalyzed by the pyruvate dehydrogenase complex
– Requires 5 coenzymes
– TPP (thiamine pyrophosphate), lipoyllysine, and FAD (flavin adenine
dinucleotide) are prosthetic groups
– NAD+ and CoA-SH are co-substrates
Structure of Coenzyme A
• Coenzymes are not a permanent part of the enzymes’ structure.
– They associate, fulfill a function, and dissociate
• The function of CoA is to accept and carry acetyl groups
Structure of Lipoyllysine
• Prosthetic groups are strongly bound to the protein
– The lipoic acid is covalently linked to the enzyme via a lysine residue
Pyruvate Dehydrogenase Complex
(PDC)
• PDC is a large (up to 10 MDa) multienzyme complex
- pyruvate dehydrogenase (E1)
- dihydrolipoyl transacetylase (E2)
- dihydrolipoyl dehydrogenase (E3)
• Advantages of multienzyme complexes:
‒ short distance between catalytic sites allows channeling of
substrates from one catalytic site to another
‒ channeling minimizes side reactions
‒ regulation of activity of one subunit affects the entire complex
Cryo-electron-microscopy of PDC
• Samples are in near-native
frozen hydrated state
• Low temperature protects
biological specimens against
radiation damage
• Electrons have smaller de Broglie
wavelength and produce much
higher resolution images than
light
3D Reconstruction from Cryo-EM data
E2 domain
Overall Reaction of PDC
Step1 and Step2 Reactions of PDC
In step 1 pyruvate reacts with the
bound thiamine pyrophosphate (TPP)
of pyruvate dehydrogenase (E1),
undergoing decarboxylation to the
hydroxyethyl derivative.
Pyruvate dehydrogenase also carries
out step 2, the transfer of two
electrons and the acetyl group from
TPP to the oxidized form of the
lipoyllysyl group of the core enzyme,
dihydrolipoyl transacetylase (E2), to
form the acetyl thioester of the
reduced lipoyl group.
Step3, Step4, Step5 Reactions of PDC
Step 3 is a transesterification in which the —
SH group of CoA replaces the —SH group of
E2 to yield acetyl-CoA and the fully reduced
(dithiol) form of the lipoyl group.
In step 4 dihydrolipoyl dehydrogenase (E3)
promotes transfer of two hydrogen atoms
from the reduced lipoyl groups of E2 to the
FAD prosthetic group of E3, restoring the
oxidized form of the lipoyllysyl group of E2.
In step 5 the reduced FADH2 of E3 transfers a
hydride ion to NAD+, forming NADH. The
enzyme complex is now ready for another
catalytic cycle.
Overall Reaction of PDC
Sequence of Events in
Oxidative Decarboxylation of Pyruvate
Enzyme 1
• Step 1: Decarboxylation of pyruvate to a hydroxyethyl
• Step 2: Oxidation of hydroxyethyl to an ester
‒ Electrons reduce lipoamide and form a thioester
Enzyme 2
• Step 3: Formation of acetyl-CoA (product 1)
Enzyme 3
• Step 4: Reoxidation of the lipoamide cofactor
• Step 5: Regeneration of the oxidized FAD cofactor
‒ Forming NADH (product 2)
Pyruvate Dehydrogenase Complex
E1
E2
E3
• E1 uses thiamine pyrophosphate
to extract carbon dioxide from
pyruvate.
• The little carrier domains of E2
need lipoic acid to hold tightly to
the acetyl groups that are
produced by the first enzyme, and
they ultimately transfer them to
another unusual chemical carrier
molecule: coenzyme A.
• The E3 enzyme that performs the
last step requires FAD and NAD
to perform a restorative reaction
on the lipoic acid.
• To accomplish these tasks, our
cells need: thiamine (vitamin B1),
pantothenic acid (B5), riboflavin
(B2), and niacin (B3) in our diets.
Number of Enzyme Units in the Complex
Enzymes
Unit
Cofactors
# subunits
prokaryotes
# subunits
eukaryotes
Pyruvate
dehydrogenase
E1
TPP (thiamine
pyrophosphate)
24
30
Dihydrolipoyl
transacetylase
E2
lipoate
coenzyme A
24
60
Dihydrolipoyl
dehydrogenase
E3
FAD
NAD+
12
12
Group Discussion: What is the role of the
multienzyme Pyruvate Dehydrogenase Complex?
Enzyme 1 can easily transfer the
Acetyl group to Enzyme 2.
Enzyme 3 can help oxidize the
reduced lipoyllysine of Enzyme
2 to its oxidized form ready for
next production step of AcetylCoA.
Enzyme 2 has the flexible
lipoyllysine group that can
switch between E1 and E3,
helped by proximity in the
ternary complex.
The Citric Acid Cycle (Krebs Cycle)
Szent-Gyorgyi
• succinate to fumarate to
malate to oxaloacetate
Martius and Knoop
• citrate to α-ketoglutarate
to succinate.
“The History of the Tricarboxylic Acid Cycle”, H.A. Krebs (1970);
Perspectives in Biology and Medicine, 14: 154-170.
Krebs
• citrate from oxaloacetate
and pyruvate, the 'missing
link‘
• In the presence of
fumarate, malate, or
oxaloacetate, succinate
also accumulated, clearly
establishing a cyclic
sequence leading to
succinate.
Sequence of Events in the Citric Acid Cycle
• Step 1: C-C bond formation to make citrate
• Step 2: Isomerization via dehydration/rehydration
• Steps 3–4: Oxidative decarboxylations to give 2 NADH
• Step 5: Substrate-level phosphorylation to give GTP
• Step 6: Dehydrogenation to give reduced FADH2
• Step 7: Hydration
• Step 8: Dehydrogenation to give NADH
Step1: C-C Bond Formation by
Condensation of Acetyl-CoA and
Oxaloacetate
Citrate Synthase
• Condensation of acetyl-CoA and oxaloacetate
• The only reaction with C-C bond formation
• Uses Acid/Base Catalysis
– Carbonyl of oxaloacetate is a good electrophile
– Methyl of acetyl-CoA is not a good nucleophile…
– …unless activated by deprotonation
• Rate-limiting step of CAC
• Activity largely depends on [oxaloacetate]
• Highly thermodynamically favorable/irreversible
– Regulated by substrate availability and product inhibition
Induced Fit in the Citrate Synthase
• Conformational change occurs upon binding oxaloacetate
• Avoids unnecessary hydrolysis of thioester in acetyl-CoA
a) Open conformation:
Free enzyme does not have a binding site for acetyl-CoA
b) Closed conformation:
Binding of OAA creates binding for acetyl-CoA
Reactive carbanion is protected
Induced Fit in the Citrate Synthase
Mechanism of Citrate Synthase
Mechanism of Citrate Synthase:
Acid/Base Catalysis
Mechanism of Citrate Synthase:
Acid/Base Catalysis
Mechanism of Citrate Synthase:
Hydrolysis of Thioester
Step2: Isomerization by
Dehydration/Rehydration
Aconitase
• Elimination of H2O from citrate gives a cis C=C bond
– Lyase
• Citrate, a tertiary alcohol, is a poor substrate for oxidation
• Isocitrate, a secondary alcohol, is a good substrate for oxidation
• Addition of H2O to cis-aconitate is stereospecific
• Thermodynamically unfavorable/reversible
– Product concentration kept low to pull forward
Iron-Sulfur Center in Aconitase
Water removal from citrate and subsequent addition to cis-aconitate
are catalyzed by the iron-sulfur center: sensitive to oxidative stress.
Aconitase is stereospecific
Only D-isocitrate is produced by aconitase.
Distinguished by three-point attachment to the active site
Group Discussion
D-Isocitrate is produced in this step, whether we
start with L-Citrate or D-Cirtate. Provide two reasons.
L-Citrate or D-Citrate
D-Isocitrate
The Citric Acid Cycle (Krebs Cycle)
Szent-Gyorgyi
• succinate to fumarate to
malate to oxaloacetate
Martius and Knoop
• citrate to α-ketoglutarate
to succinate.
“The History of the Tricarboxylic Acid Cycle”, H.A. Krebs (1970);
Perspectives in Biology and Medicine, 14: 154-170.
Krebs
• citrate from oxaloacetate
and pyruvate, the 'missing
link‘
• In the presence of
fumarate, malate, or
oxaloacetate, succinate
also accumulated, clearly
establishing a cyclic
sequence leading to
succinate.
Step3: Oxidative Decarboxylation #2
Isocitrate Dehydrogenase
• Oxidative decarboxylation
– Lose a carbon as CO2
– Generate NADH
• Oxidation of the alcohol to a ketone
– Transfers a hydride to NAD
• Cytosolic isozyme uses NADP+ as a cofactor
• Highly thermodynamically favorable/irreversible
– Regulated by product inhibition and ATP
Mechanisms of Isocitrate Dehydrogenase
Mechanisms of Isocitrate Dehydrogenase:
Metal Ion Catalysis (Oxidation)
Mechanisms of Isocitrate Dehydrogenase:
Metal Ion Catalysis (Decarboxylation)
Carbon lost as CO2 did NOT come from acetyl-CoA.
Mechanisms of Isocitrate Dehydrogenase:
Rearrangement and Product Release
Mechanisms of Isocitrate Dehydrogenase
Biological Chiral Recognition: A
Substrate's Perspective;
Sundaresan V, Abrol R (2005).
Chirality, 17(Suppl):S30-9.
Towards a general model for proteinsubstrate stereoselectivity;
Sundaresan V, Abrol R (2002).
Protein Sci, 11(6): 1330-9.
Structural biology: A new model for protein
stereospecificity
Andrew D. Mesecar1 & Daniel E. Koshland,
Jr, Nature 403, 614-615 (10 February 2000)
Step4: Final Oxidative Decarboxylation
α-Ketoglutarate Dehydrogenase
• Last oxidative decarboxylation
– Net full oxidation of all carbons of glucose
• After two turns of the cycle
• Carbons not directly from glucose because carbons lost came from
oxaloacetate
• Succinyl-CoA is another higher-energy thioester
bond
• Highly thermodynamically favorable/irreversible
– Regulated by product inhibition
Origin of C-atoms in CO2
COOH
H2C
COOH
C
COOH
HC
COOH
H2C
COOH
C
H
COOH
H2C
HO
Citrate
HO
Isocitrate
H2C
COOH
H2C
CH2
CH2
O
C
COOH
α-ketoglutarate
COOH
O
C
SCoA
Succinyl-CoA
Both CO2 carbon atoms derived from oxaloacetate
α-Ketoglutarate Dehydrogenase
• Complex similar to pyruvate dehydrogenase
– Same coenzymes, identical mechanisms
– Active sites different to accommodate different-sized substrates
Step5: Generation of GTP through Thioester
Succinyl-CoA Synthetase
• Substrate level phosphorylation
• Energy of thioester allows for incorporation of
inorganic phosphate
• Goes through a phospho-enzyme intermediate
• Produces GTP, which can be converted to ATP
• Slightly thermodynamically favorable/reversible
– Product concentration kept low to pull forward
Mechanism of Succinyl-CoA Synthetase
Step6: Oxidation of an Alkane to Alkene
Succinate Dehydrogenase
• Bound to mitochondrial inner membrane
– Part of Complex II in the electron-transport chain
• Reduction of the alkane to alkene requires FADH2
– Reduction potential of NAD is too low
• FAD is covalently bound, unusual
• Near equilibrium/reversible
– Product concentration kept low to pull forward
Step7: Hydration Across a Double Bond
Fumarase
• Stereospecific
– Addition of water is always trans and forms L-malate
– OH- adds to fumarate… then H+ adds to the carbanion
– Cannot distinguish between inner carbons, so either can
gain –OH
• Slightly thermodynamically favorable/reversible
– Product concentration kept low to pull reaction forward
Stereospecificity of Fumarase
Step8: Oxidation of Alcohol to a Ketone
Malate Dehydrogenase
• Final step of the cycle
• Regenerates oxaloacetate for citrate synthase
• Highly thermodynamically UNfavorable/reversible
– Oxaloacetate concentration kept VERY low by citrate synthase
• Pulls the reaction forward
One Turn of the Citric Acid Cycle
Net Result of the Citric Acid Cycle
Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O 
2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
• Net oxidation of two carbons to CO2
– Equivalent to two carbons of acetyl-CoA
– but NOT the exact same carbons
• Energy captured by electron transfer to NADH and
FADH2
• Generates 1 GTP, which can be converted to ATP
• Completion of cycle
Direct and Indirect ATP Yield
30 x (-50kJ/mol) ~ -1500 kJ/mol
>50% efficiency based on glucose energy of -2840 kJ/mol
Efficiency of gasoline based motor engines: ~20%
Regulation of the Citric Acid Cycle
Group Discussion
Mechanisms of regulation?
Regulation of the Citric Acid Cycle
• Regulated at highly thermodynamically favorable
and irreversible steps
– PDH, citrate synthase, IDH, and KDH
• General regulatory mechanism
– Activated by substrate availability
– Inhibited by product accumulation
– Overall products of the pathway are NADH and ATP
• Affect all regulated enzymes in the cycle
• Inhibitors: NADH and ATP
• Activators: NAD+ and AMP
Regulation of Pyruvate Dehydrogenase
• Also regulated by reversible phosphorylation of E1
– Phosphorylation: inactive
– Dephosphorylation: active
• PDH kinase and PDH phosphorylase are part of
mammalian PDH complex
– Kinase is activated by ATP
• High ATP  phosphorylated PDH  less acetyl-CoA
• Low ATP  kinase is less active and phosphorylase
removes phosphate from PDH  more acetyl-CoA
Phosphorylation and its use in regulation in Cellular Signaling
to be discussed in lectures next week.
Additional Regulatory Mechanisms
• Citrate synthase is also inhibited by succinyl-CoA
– α-ketoglutarate is an important branch point for amino
acid metabolism
– Succinyl-CoA communicates flow at this branch point to
the start of the cycle
• Regulation of isocitrate dehydrogenase controls
citrate levels
– Aconitase is reversible
– Inhibition of IDH leads to accumulation of isocitrate and
reverses acconitase
– Accumulated citrate leaves mitochondria and inhibits
phosphofructokinase in glycolysis
Glyoxylate Cycle
Glyoxylate Cycle
• Found in plants and some microorganisms
• Net production of 2 acetyl-CoA  oxaloacetate
– Allows net conversion of acetyl-CoA to glucose, which
animals cannot accomplish
• Compartmentalized in the glyoxysome
– Part of the citric acid cycle
– Bypasses the decarboxylation with two different enzymes
• Isocitrate lyase
• Malate synthase
Compartmentation of Glyoxylate Cycle
Chapter 16: Summary
In this chapter, we learned:
• A large multi-subunit enzyme, pyruvate dehydrogenase complex,
converts pyruvate into acetyl-CoA
• Several cofactors are involved in reactions that harness the
energy from pyruvate
• Citric acid cycle is an important catabolic process: it makes GTP
and reduced cofactors that could yield ATP
• Citric acid cycle plays important anabolic roles in the cell
• Organisms have multiple ways to replenish intermediates that
are used in other pathways
• The rules of organic chemistry help to rationalize reactions in the
citric acid cycle
• The citric acid cycle is largely regulated by availability of
substrates and product inhibition (especially NADH and ATP)