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Biochemistry I
Citric Acid Cycle
Dehydrogenases
and Cycle Overview
Chapter 17 – part 1
Dr. Ray
Textbook Sections: 17.1, 17.2
Oxidative Fuel Metabolism
Pyruvate Dehydrogenase
Complex
(1) Glycolysis: conversion of one 6C glucose to two 3C pyruvate, with energy output of:
• 2 ATP (substrate level phosphorylation)• 2 NADH (carriers of high energy electrons)
(2) Pyruvate Dehydrogenase Complex:(PDHC in mitochondria) converts 3C pyruvate to 2C acetyl CoA with loss of 1C carbon dioxide via a decarboxylation reaction
(3) Citric Acid Cycle:
of enzymatic reactions for the complete oxidation of 2C acetyl groups to two one-carbon CO2 molecules
• Has multiple C-C bond breaking and forming steps
• Also called the tricarboxylic acid (TCA) cycle or the Krebs cycle
The citric acid cycle is the final common pathway for the oxidation of fuel molecules: amino acids, fatty acids, and carbohydrates, all enter the cycle as acetyl Coenzyme A.
is a cyclic system
occurs in cytosol
occur in mitochondria
__________ is transported
into mitochondria
Pyruvate
C
C
CH3
O O-
O C
S
CH3
O
CoA
Pyruvate Acetyl CoA
+ CO2
Oxidative a-decarboxylation
• Mitochondria has a double membrane
• Invaginations of inner mitochondrial membrane (IMM)are CRISTAE
• Glycolysis – occurs in cytoplasm (product pyruvate is transported into mitochondria)
• PDHC and Citric Acid cycle reactions – occur in the lumen (interior space or MATRIX of mitochondria)
• Electron Transport and Oxidative Phosphorylation– occurs in proteins imbedded in the IMM
Pyruvate Dehydrogenase Complex (PDHC)
PDHC – connects glycolysis & Citric Acid Cycle pathways
Pyruvate
enter Electron
Transport Chain
Electrons have high reduction potential:
Mitochondria: Energy Powerhouse of Cells• Electron Transport Chain and Oxidative Phosphorylation
– occurs within proteins imbedded in inner mitochondrial membrane:
• In the electron transport chain, electrons from NADH flow through various proteins with O2 is the final electron acceptor.
• The oxidation of NADH &FADH2 releases energy, and this energy powers the formation of proton gradient: so H+ ions are pumped across the inner mitochondrial membrane!
• These proton gradients can in turn drive the synthesis of ATPwhen H+ ions flow through the ATP synthase back into the mitochondria.
electronsTCA
oxidative phosphorylation
Aerobic electron transfer & phosphorylation are energetically coupled
have reduction potential (high energy electrons)
C
C
CH3
O O-
O C
S
CH3
O
CoA
Pyruvate Acetyl CoA
+ CO2
Oxidative a-decarboxylation
Redox Balance in Aerobic Metabolism
Pyruvate Dehydrogenase Complex (PDHC) contains 3 enzymes and 5 cofactors(do NOT need to know the details of this mechanism, only overall reaction)
http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/animations/animations.htm PDHC Intro
• In oxidative metabolism, pyruvate
is converted to ____________ __ by
the action of the PDHC, which then
enters the citric acid cycle.
PDHC
PDHC: Entry Port for Citric Acid Cycle
C
C
CH3
O O-
O C
S
CH3
O
CoA
Pyruvate Acetyl CoA
+ CO2
The entry port to this oxidative pathway is the formation of acetyl CoA from pyruvate, catalyzed by the
Pyruvate + NAD+ + CoA Acetyl CoA + CO2 + NADH
1. Hydrolysis of what functional group releases the energy in Acetyl CoA?
2. How many, and which carbon(s) of pyruvate are oxidized in this step?
3. This is an ___________________________ reaction, since the C-C bond broken is between the carbonyl (C2) and Ca (C1) 4. How is the energy released by oxidation
of C1 and C2 captured?
oxidative a-decarboxylation
1
2
3
Decarboxylation & OxidationOxidation
a to C=O
CoASH + (CoA)
NAD+ + + NADH
pyruvate dehydrogenase complex (PDHC)
Two
energetically coupled to formation of thioester
(1) NADH ( __________ potential)(2) Acetyl CoA ( ______ transfer potential) so it is a ______________ compound
C1 _________________C2 _________________C3 __________
Acetyl CoA + H2O Acetate + CoASH
DGo’ = - 31.4 kJ/mol
• Large complex multisubunit structure with 3 highly integrated enzymes
Pyruvate Dehydrogenase Complex (PDHC):
Multimolecular Aggregate
CoASH stoichiometric cosubstrate
NAD+ stoichiometric cosubstrate
Pyruvate + CoA + NAD+ Acetyl CoA + CO2 + NADH + H+
Do NOT need to know PDHC mechanism
• Contains 5 cofactors (3 prosthetic groups & 2 cosubstrates)
• Catalyzes a complex a-decarboxylation reaction
Acetyl CoA: Activated Carrier of 2-Carbon Units
The activated carrier of two-carbon units is Acetyl-Coenzyme A, a metabolic intermediate that is central to many pathways, such as the citric acid cycle. Pyruvate is converted to Acetyl CoA by the ___________________________________________ .
• Acetyl CoA has an acyl group linked to organic Coenzyme A.
• The DG°’ for the hydrolysis of acetyl CoA has a large negative value exergonic reaction
• Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group:
?
Thioester bonds have high (acetyl) group transfer potential, which can be released by hydrolysis
Pyruvate Dehydrogenase Complex (PDHC)
Acetyl CoA + H2O Acetate + CoASH DGo’ = - 31.4 kJ/mol
Citric Acid Cycle: Amphibolic PathwayThe citric acid cycle is the central metabolic hub of the cell:• It is the gateway to the aerobic metabolism of any molecule that can be
transformed into an acetyl group or a dicarboxylic acid.
• It is also an important source of precursors forming the building blocks of many other molecules such as amino acids, nucleotide bases, cholesterol, and porphyrin (the organic component of heme), and providing metabolic intermediates for anabolism (biosynthesis).
• The citric acid cycle includes a series of four oxidation-reduction reactions, in which the
organic species are _________ and four cofactors are ____________ .
C
S
CH3
O
CoA
Acetyl CoA
2 CO2
http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/animations/animations.htmCitric Acid Cycle Carbon
Acetyl CoA + 3 NAD+ + FAD + Pi + GDP
2 CO2 + CoASH + 3 NADH + FADH2 + GTP
Citric Acid Cycle
1) Acetyl CoA (2C) joins with oxaloacetate (4C) to form citrate (6C)
2) 2 decarboxylation steps occur, with regeneration of 4C oxaloacetate and output of 2 CO2 molecules
3) Four oxidation steps occur which produce3 NADH and 1 FADH2
4) 1 GTP made by Substrate Level Phosphorylation
• Much more energy is extracted aerobically by means of the citric acid cycle and the mitochondrial electron transport chain, than is extracted by anerobic formation of lactate.
• Oxidative Phosphorylation requires _______ as final electron acceptor, and traps metabolic reducing energy in the form of _________ .
In one turn of the citric acid cycle, the following occur:
Overall: One acetyl CoA is converted to two molecules of CO2 :
Stages of catabolism:
Oxidative (Aerobic) Fuel Metabolism
cyclic system
H
CHO
OH
HHO
OHH
OHH
CH2OH
C
C
CH3
O O-
O C
S
CH3
O
CoA
CO2
1. In order to convert 1 glucose into 6 CO2, how many C-C bonds need to be broken?
Number of C-C bonds BROKEN:
Number of C-C bondsFORMED:
NET ___ C-C bonds in glucose (C6H12O6) are broken, mostly by
_________________________ reactions
1 glucose 2 pyruvate 2 acetyl CoA 4 CO2
+ 2 CO2
CO2
(4C) (6C) (5C)
(4C)(4C)
Acetyl CoA(2C)
CO2
Glycolysis PDHC Citric Acid Cycle
2. What bonds are formed & broken?
Metabolites in Citric Acid Cycle Phase I: one C-C bond formation and two decarboxylations:
Phase II: Regenerate Oxaloacetate:
Fumarate (4C) Malate (4C) Oxaloacetate (4C)
• Nomenclature of simple dicarboxylic acid metabolic intermediates is based on the number of carbon atoms present in the linear chain:
5C Glutarate 4C Succinate 3C Malonate
Oxaloacetate (4C) Citrate (6C) a-Ketoglutarate (5C)
Succinyl-CoA (4C) Succinate (4C)
+ GTP
Acetyl CoA
Glutarate (5C dicarboxylic acid) with a ketone alpha to one of the carboxylates
For all 8 reactions you need to know:
1. Metabolite structures 2. Type of reaction 3. Name of enzyme catalyst 4. Cofactors and small
molecules involved5. Energy output6. Mechanism
- citrate synthase (step 1)- isocitrate dehydrogenase
(step 3)7. Energetics & Mechanism
- succinyl CoA synthase (5)
Phase IPhase II
• A cyclic system of enzymatic reactions that result in oxidation of 2C acetyl CoA to CO2 with concomitant production of reduced coenzymes
•Remove CO2& extract
energy•Regenerate oxaloacetate
http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/animations/animations.htm Citric Acid Cycle Energy
Summary of the Citric Acid Cycle
Voet, Voet & Pratt, Fundamentals of
Biochemistry, Fig. 16.2
The Citric Acid Cycle
1
3
2
5
4
6
7
For one turn of cycle:
In:
• 1 Acetyl CoA
Out:
• 2 CO2
• 3 NADH
• 1 FADH2
• 1 GTP
8
1 Glucose
2 Pyruvate
2 Acetyl-CoA
4 CO2
2 CO2
1. How many turns of TCA cycle are needed per glucose?
2. Can these happen at the same time?
____ have multiple copies of all catalytic metabolic enzymes
DEHYDROGENASES (oxidoreductases) catalyze several types of reactions:
1. Loss of a pair of H atoms (to form alkene)
2. Oxidation of 2o alcohol to ketone
3. Oxidation of aldehyde to carboxylic acid (or its acyl phosphate derivative)
4. Oxidation of 2o alcohol to ketone,coupled to b-decarboxylation
5. Oxidation of ketone to a carboxylic acid (or its thioester derivative) coupled to a-decarboxylation
Dehydrogenases in Glucose Catabolism:
Redox reactions that form NADH and FADH2
1. Take each dehydrogenase reaction given in the list below, and determine which step in complete glucose catabolism (1 glucose 6 CO2) involves this type of dehydrogenase function.
Dehydrogenases are enzymes that catalyze oxidation reactions of metabolic intermediates while reducing NAD+ to NADH or FAD to FADH2 .
Answer using HANDOUT
(next slide)
Voet, Voet & Pratt, Fundamentals of Biochemistry, Fig. 17.1
1
3
2
5
4
6
7
8
1
2
3
4
5
COO-
C
CH3
O
S-CoA
C
CH3
O
(a) (d)
Pyruvate Dehydrogenase Complex
+ CO2
The Citric Acid Cycle
Glycolysis
C
OHH
CH2 O P
O
O-
O-
O HC
OHH
CH2 O P
O
O-
O-
O OP
O
O-O-
GAP 13BPG
Glyceraldehyde-3-P Dehydrogenase
NADH
NAD+
+ Pi
1 turn of cycle:In: • 1 Acetyl CoA
Out:• 2 CO2
• 3 NADH
• 1 FADH2
• 1 GTP
1 Glucose 2 turns of TCA
6
SPECIFIC DEHYDROGENASE (DH) FUNCTIONSDehydrogenases are enzymes that oxidize metabolic intermediates
and reduce NAD+ to NADH or FAD to FADH2, thus helping to: CAPTURE the free energy released during
oxidative glucose catabolism!
pyruvate DH
isocitrate DH
and a-ketoglutarate DH
DH-1• Some involve simple oxidations: succinate DH and malate DH
• Some involve Oxidative Decarboxylatons, which comes in two flavors:
• a-decarboxylation:
• b-decarboxylation:
• Some involve oxidations that are coupled to forming a high-energy
intermediate whose hydrolysis in the next step provides the energy for either:
(i) Glyceraldehyde DH reaction to form 1,3-BPG in glycolysis
(i) a-ketoglutarate DH reaction to form Succinyl CoA in TCA
(ii) Pyruvate DH reaction to form Acetyl CoA(links glycolysis & TCA)
DH-4
DH-5
DH-3
DH-5
DH-5
DH-2
(i) substrate level phosphorylation or
• Some involve oxidations that are coupled to forming a high-energy intermediate whose hydrolysis in the next step provides the energy for either: (ii) C-C bond formation
http://higheredbcs.wiley.com/legacy/college/voet/0471214957/guided_ex/citric_acid_cycle/citric_acid_cycle.html
Use ATP count used in Stryer text: 1 NADHM = 2.5 ATP, 1 FADH = 1.5 ATP
P/O ratio:
Interactive TCA website
Citric Acid Cycle
COO-
CH2
C OH-OOC
CH2
COO-
COO-
C
C H-OOC
CH2
COO-
H OH
COO-
C
C HC
CH2
COO-
O
COO-
C
C HH
CH2
COO-
O
COO-
C
CH2
CH2
COO-
O
S
C
CH2
CH2
COO-
O
CoA
O-
C
CH2
CH2
COO-
O COO-
CH2
CH2
COO-
COO-
CH2
CH2
COO-
COO-
C
CH2
COO-
HO HC
C
COO-H
-OOC H
COO-
C
CH2
COO-
O
COO-
CH2
C OH-OOC
CH2
CO-O
COO-
CH2
C-OOC
O
CH3
CSO CoA
CO2
CO2
CoASH
GDP + PiCoASH
H2OCOO-
CH2
C-OOC
O
CoASH
H2O+
Citrate Isocitrate a-Ketoglutarate
Oxalosuccinate
Oxaloacetate
Acetyl CoA
a-Ketoglutarate
Succinate Fumarate Malate Oxaloacetate
=
=
Succinyl CoA Succinate
GTP +
=
O
-O
4 5
1 2 3
6 7 8
[3b][3a]
1
2
3
4
5
rotate OAA
NADH
NADH
FADH2 NADH
6
Q: Name each enzyme in TCA?
aKGDH reaction is similar to PHDC rxn:
Using energy from hydrolysis
Citric Acid Cycle
COO-
CH2
C OH-OOC
CH2
COO-
COO-
C
C H-OOC
CH2
COO-
H OH
COO-
C
C HC
CH2
COO-
O
COO-
C
C HH
CH2
COO-
O
COO-
C
CH2
CH2
COO-
O
S
C
CH2
CH2
COO-
O
CoA
O-
C
CH2
CH2
COO-
O COO-
CH2
CH2
COO-
COO-
CH2
CH2
COO-
COO-
C
CH2
COO-
HO HC
C
COO-H
-OOC H
COO-
C
CH2
COO-
O
COO-
CH2
C OH-OOC
CH2
CO-O
COO-
CH2
C-OOC
O
CH3
CSO CoA
CO2
CO2
CoASH
GDP + PiCoASH
H2OCOO-
CH2
C-OOC
O
CoASH
H2O+
Citrate Isocitrate a-Ketoglutarate
Oxalosuccinate
Oxaloacetate
Acetyl CoA
a-Ketoglutarate
Succinate Fumarate Malate Oxaloacetate
=
=
Succinyl CoA Succinate
GTP +
=
O
-O
4 5
substrate level phosphorylation
1 2 3
6 7 8
oxidative b-decarboxylation
oxidative a-decarboxylation
[3b][3a]
1
2
3
4
5
C-C bond formation (Aldol condensation)
isomerization
rotate OAA
oxidation 2oROH
ketone
oxidation oxidation hydration
NADH
NADH
FADH2 NADH
6
The two CO2 that come off during one round of the cycle: are NOT the two carbons that go in as Acetyl CoA in that round, instead they come from oxaloacetate
Isomerization by ACONITASE always moves the central alcohol
AWAY from acetyl-CoA side
1
3
2
5
4
6
7
8
1
2
3
4
5
COO-
C
CH3
O
S-CoA
C
CH3
O
(a) (d)
Pyruvate Dehydrogenase Complex
+ CO2
5
4
The Citric Acid Cycle
The two CO2 that come off during one round of the cycle are NOT the two that go in as Acetyl CoA in that round
DH-1
DH-2
DH-5
DH-4
DH-5
DH-3
Glycolysis
C
OHH
CH2 O P
O
O-
O-
O HC
OHH
CH2 O P
O
O-
O-
O OP
O
O-O-
GAP 13BPG
Glyceraldehyde-3-P Dehydrogenase
NADH
NAD+
+ Pi
1 turn of cycle:In: • 1 Acetyl CoA
Out:• 2 CO2
• 3 NADH
• 1 FADH2
• 1 GTP
1 Glucose 2 turns of TCA
6
The second CO2 lost comes from C4 of OAA
The first CO2 lost comes from the C1 at the top of OAA
The Citric Acid Cycle Problems:1) Malonate anion is a potent competitve inhibitor of succinate dehydrogenase, which catalyzes the conversion of succinate to fumarate
(a) Why is malonate unreactive?
COO
CH2
CH2
COO
COO
CH2
COO
Succinate Malonate
Substrate Comp. Inhibitor
2) How many metabolites in the citric acid cycle are tricarboxylic acids?(A) 1 (B) 2 (C) 4(D) 6 (E) 8
Succinate has __ methylene groups, which loses ___ hydrogens during oxidation. Malonate has __ methylene group and cannot be dehydrogenated, so is unreactive.
3) How many are dicarboxylic acids?
4) Oxidation of one mole of acetyl-CoA via the citric acid cycle results in NET (compared made to used in one turn):
A) production of one mole of citrateB) consumption of one mole of oxaloacetateC) production of 7 moles of ATPD) production of one mole of succinateE) production of 2 moles of CO2
Citric Acid Cycle Reactions
The three reactions with the largest driving force (DGo’) are:• Citrate Synthase (C-C bond formation)
• Isocitrate Dehydrogenase (b-decarboxylation)
• a-Ketoglutarate Dehydrogenase (a-decarboxylation)
- hydrolysis of thioester
- oxidation
- oxidation
Standard conditions
Step 1: Citrate Synthase C-C bond formation
4C + 2C 6C
Oxaloacetate + Acetyl CoA Citrate + CoASH
• One of few enzymes that can form a C-C bond without assistance of a
metal ion cofactor. An aldol condensation reaction forms the C-C bond.
1. How many carboxylic acid groups in:
(a) citrate?
(b) oxaloacetate?
(c) acetyl CoA?
3. Which reactant (functional group) is the nucleophile and which is the electrophile in this rxn?
Acetyl CoA
Oxaloacetate
4. The center C of citrate is a ___ alcohol
5. What functional groups react, and what product is formed?
1
2
3
4
+
Ketone
Ketone
b-hydroxy-acid a carboxylic acid derivative that can hydrolyze
to RCO2-
a
b
The citric acid cycle begins with the condensation of a 4-carbon unit, oxaloacetate, and a 2-carbon unit, the acetyl group of acetyl CoA.
• The reaction catalyzed by citrate synthase is an (A) aldol condensationfollowed by a hydrolysis. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then (B) hydrolyzed to citrate and CoA.
• The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate.
• The hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.
Step 1: Citrate Synthase C-C bond formation
A B
Because this reaction initiates the Citric Acid Cycle, it is very important that
SIDE REACTIONS BE MINIMIZED!
Step 1: Citrate Synthase C-C bond formation
4C + 2C 6C
Oxaloacetate + Acetyl CoA Citrate + CoASH
• Bisubstrate enzyme – binding of first substrate (oxaloacetate) results in
large 18o rotation of subunits in dimer to form the binding site for the
second substrate (acetyl CoA), via an
• Binding of oxaloacetate induces a major structural rearrangement
leading to the creation of a binding site for acetyl CoA (2nd substrate).
• Open form of the enzyme occurs in the absence of ligands, and is con-
verted into a closed form by the binding of the 1st substrate oxaloacetate.
(a) open conformation (b) closed conformation
+ oxalo-acetate
H2O +
• Trisubstrate
ordered sequential mechanism.
third substrate
Step 1: Citrate Synthase Mechanism1. First substrate oxaloacetate (OAA) binds to Citrate Synthase enzyme
2. Enzyme deprotonates acetyl CoA to form carbanion/enolate via acid-base catalysis: Asp375 ( ____ catalyst) and His274 (_____ catalyst)
3. Aldol Condensation Reaction (to form C-C bond):
• Nucleophilic enol attacks carbonyl C of oxaloacetate (electrophile).
• His320 protonates resulting tetrahedral oxyanion to form an alcohol.
• Thus C-C bond formation occurs, and the citryl is still attached to CoAvia thioester linkage (Citryl CoA complex)
2
Cytrate
synthase
+ OAA
1
2nd conformation change:occurs after C-C bond formation- now forms hydrolysis site
3
Nucleophile
Electrophile
1st conforma-tion change:occurs after OAA binds to enzyme - now can bind 2nd acetyl CoA substrate
C=O
His 320
So can bind _______________
4. Hydrolysis of “high energy” thioester citryl CoA (electrophile) by
nucleophile H2O, forms carboxylate (deprotonated carboxylic acid) and
thiol (CoASH = Coenzyme A).
• This is an exergonic reaction that pulls the previous two equilibrium
steps (2,3) to the right, allowing formation of the final product, 6C citrate.
Step 1: Citrate Synthase Mechanism
His 274
OH2
4
1) Which bond is hydrolyzed?
• His 274 now participates as a proton donor to
stabilize the oxianion. Then the C=O bond
reforms with CoASH as the leaving group.
• Coenzyme A (CoASH) leaves the enzyme,
followed by citrate, and the enzyme returns to
the initial open conformation.
2) How many thioester species are involved in this reaction?
Mechanism of Citrate Synthase Oxaloacetate + Acetyl CoA + H2O Citrate + CoASH
(A)
(C)
(E)
(B)
(D)
(F)
Q: In what order do these steps occur?
1st conforma-tion change:
occurs after OAA binds to enzyme
now can bind 2nd acetyl CoA substrate
2nd conforma-tion change:
occurs after C-C bond formation
now forms hydrolysis site and binds H2O
Lehninger – Principles of Biochemistry5th Ed, animations (chapter 16), by Nelson and Cox, 2008 W. H. Freeman & Company
Citrate synthase catalyzes condensation reaction by (mechanism):
• Bringing substrates into _______________________________________ ______________ certain bonds
• Using general _________________________
• The hydrolysis of the _____________ powers the synthesis of a new molecule from 2 precursors (C-C bond formation)
1. How is the wasteful hydrolysis of acetyl CoA prevented (since this also has a high-energy thioester linkage)?
a. Acetyl CoA does NOT bind to the enzyme until after oxaloacetate is bound and ready for condensation.
b. The catalytic residues crucial for hydrolysis of the thioester linkage are not appropriately positioned until citryl CoA is formed.
c. Formation of the citryl CoA intermediate induces additional structural changes in the enzyme, so that the active site becomes completely enclosed.
Step 1: Citrate Synthase Mechanism – Summary
As with hexokinase and triose phosphate isomerase, induced fit conformational changes prevent undesirable side reactions(hydrolysis of high energy species with loss of energy)
