Download Citric Acid Cycle

Citric Acid Cycle (CAC)
AKA:
Tricarboxylic Acid (TCA) Cycle
and Krebs Cycle
After this Lecture you will
be able to answer:
• For each step of the TCA cycle:
– What do we get out of this?
– Thermodynamics?
– Is it Regulated? How?
• Where do the carbons of Glucose become fully
oxidized?
• Why is the TCA cycle amphibolic?
• How are TCA cycle intermediates replenished?
Summary of Citric Acid Cycle
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi
2 CO2 + 3 NADH + 3H+ + FADH2 + GTP + CoA-SH
Citric Acid Cycle
Figure 17-2
Citrate Synthase
(citrate condensing enzyme)
CoA–SH
O
H3C
C
+
S
CoA
Acetyl-SCoA
C
COOH
H2C
COOH
O
Oxaloacetate
∆Go’ = –31.5 kJ/mol
H2C
HO
COOH
C
COOH
H2C
COOH
Citrate
Mechanism of Citrate Synthase
(Formation of Acetyl-SCoA Enolate)
Figure 17-10 part 1
Mechanism of Citrate Synthase
(Acetyl-CoA Attack on Oxaloacetate)
Figure 17-10 part 2
Mechanism of Citrate Synthase
(Hydrolysis of Citryl-SCoA)
Figure 17-10 part 2
Regulation of Citrate Synthase
• Pacemaker Enzyme (rate-limiting step)
• Rate depends on
• Availability of substrates
– Acetyl-SCoA
– Oxaloacetate
• Reduction Potential (NAD+/NADH)
• Energy Charge (ATP/ADP/AMP)?
• Inhibited by:
– Citrate
– Succinyl-CoA
Aconitase
∆Go’ = ~0
H2C
HO
COOH
H2O
H2C
COOH
C
COOH
C
COOH
H2C
COOH
HC
COOH
Citrate
(~91%)
H2O
H2C
COOH
HC
COOH
C
H
COOH
HO
Cis-aconitate
(~3%)
Isocitrate
(~6%)
Stereospecific
Addition
Iron-Sulfur Complex
(4Fe-4S]
Thought to coordinate citrate –OH
to facilitate elimination
Stereospecificity of Aconitase Reaction
Prochiral
Substrate
Stereospecificity in
Substrate Binding
Page 325
Chiral
Product
NAD+–Dependent
Isocitrate Dehydrogenase
∆Go’ = -20.9 kJ/mol
NAD+
H2C
COOH
HC
COOH
C
H
COOH
HO
Isocitrate
NADH + H+
H2C
Mn2+ or Mg2+
COOH
CH2
O
C
COOH
-ketoglutarate
Oxidative Decarboxylation
NOTE: CO2 from oxaloacetate
+ CO2
Mechanism of
Isocitrate Dehydrogenase
(Oxidation of Isocitrate)
Figure 17-11 part 1
Mechanism of
Isocitrate Dehydrogenase
(Decarboxylation of Oxalosuccinate)
Mn2+ polarizes C=O
Figure 17-11 part 2
Mechanism of
Isocitrate Dehydrogenase
(Formation of -Ketoglutarate)
Figure 17-11 part 2
Regulation of Isocitrate Dehydrogenase
• Pulls aconitase reaction
• Regulation (allosteric enzyme)
– Energy Charge (ATP/ADP)
– Ca2+
• Competitive Inhibition
– Reduction Potential (NADH/NAD+)
• Accumulation of Citrate:
– inhibits Phosphofructokinase
Accumulation of Citrate
CO2
Isocitrate
dehydrogenase
CO2
Isocitrate
dehydrogenase
-Ketoglutarate Dehydrogenase
∆Go’ = -33.5 kJ/mol
NAD+
H2C
COOH
CH2
O
C
NADH + H+
H2C
+ CoASH
COOH
COOH
CH2
O
C
SCoA
Succinyl-CoA
-ketoglutarate
Oxidative Decarboxylation
Mechanism similar to PDH
CO2 from oxaloacetate
High energy thioester
+ CO2
-Ketoglutarate Dehydrogenase
(Multienzyme Complex)
• E1: -Ketoglutarate Dehydrogenase
or -Ketoglutarate Decarboxylase
• E2: Dihydrolipoyl Transsuccinylase
• E3: Dihydrolipoyl Dehydrogenase
(same as E3 in PDH)
Regulation of
-Ketoglutarate Dehydrogenase
• Competitive Inhibitors
– NADH
– Succinyl-SCoA
• Activator: Ca2+
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
O
C
COOH
-ketoglutarate
COOH
CH2
O
C
SCoA
Succinyl-CoA
Both CO2 carbon atoms derived from oxaloacetate
Succinyl-CoA Synthetase
∆Go’ = ~0
GDP + Pi
H2C
COOH
CH2
O
C
SCoA
Succinyl-CoA
GTP
H2C
COOH
H2C
COOH
+ CoASH
Succinate
High Energy Thioester —> Phosphoanhydride Bond
Plants and Bacteria: ADP + Pi —> ATP
Randomizationn of labeled C atoms
Thermodynamics
(Succinyl-SCoA Synthetase)
Succinyl-SCoA+ H2O
GDP + Pi
Succinyl-SCoA + GDP + Pi
Succinate + CoA
² Go' = –32.6 kJ/mol

GTP + H2O
Go' = +30.5 kJ/mol
²
Succinate + GTP + CoA
² Go'

= –2.1 kJ/mol
Mechanism of
Succinyl-CoA Synthetase
(Formation of High Energy Succinyl-P)
Figure 17-12 part 1
Mechanism of
Succinyl-CoA Synthetase
(Formation of Phosphoryl-Histidine)
Figure 17-12 part 2
Mechanism of
Succinyl-CoA Synthetase
(Phosphoryl Group Transfer)
Substrate-level phosphorylation
Figure 17-12 part 3
Nucleoside Diphosphate Kinase
(Phosphoryl Group Transfer)
GTP + ADP ——> GDP + ATP
∆Go’ = ~0
Which of the following is/are advantages
of multienzyme complexes?
A. They speed up reaction rates.
B. Side reactions are limited by channeling
C. The entire complex can be controlled as one unit.
D. A and C
E. All of the above
Predict which of the following enzymes are identical in
both the pyruvate dehydrogenase and the ketoglutarate dehydrogenase complexes.
A. E1
B. E2
C. E3
D. E2 and E3
E. E1, E2 and E3
Which of the following enzymes catalyzes a
reaction with the pictured compound as an
intermediate?
A.
B.
C.
D.
E.
a-ketoglutarate dehydrogenase
succinyl-CoA synthetase
succinate dehydrogenase
fumarase
malate dehydrogenase
Succinate Dehydrogenase
∆Go’ = ~0
FAD
H2C
COOH
H2C
COOH
Succinate
FADH2
CH
HOOC
HC
COOH
Fumarate
Membrane-Bound Enzyme
Direct entry into the ETC
Covalent Attachment of FAD
Figure 17-13
FAD used for Alkane  Alkene
• Reduction Potential (E)
– Affinity for electrons; Higher E, Higher Affinity
– Electrons transferred from lower to higher E
FAD/FADH2
Reduction
Potential
Succinate/Fumarate
NAD+/NADH
Isocitrate/α-Ketoglutarate
Fumarase
∆Go’ = ~0
H2O
HOOC
CH
HC
H
C
COOH
H2C
COOH
HO
COOH
Fumarate
Malate
Malate Dehydrogenase
NAD+
H
C
COOH
H2C
COOH
HO
NADH + H+
C
COOH
H2C
COOH
O
Oxaloacetate
Malate
∆Go’ = +29.7 kJ/mol
Low [Oxaloacetate]
Availability of NAD+
Thermodynamics
Malate + NAD+
Oxaloacetate + NADH + H+
Acetyl-SCoA + Oxaloacetate
Citrate + CoA
Malate + NAD+
NADH + H+ +
+ Acetyl-SCoA
Citrate + CoA
² Go' = +29.7 kJ/mol
² Go' = –31.5 kJ/mol

² Go'
= –1.8 kJ/mol
Products of the Citric Acid Cycle
Figure 17-14
ATP Production from Products
of the Central metabolic Pathway
= 32 ATP
NADH  2.5 ATP
FADH2 1.5 ATP
Page 584
1
6
Carbons of Glucose:
1st cycle
2
5
3
4
3, 4
2,5
1,6
1,6
2,5
2,5
1,6
1,6
2,5
2,5
1,6
Carbon Tracing from Glucose
Carbon Tracing from Glucose
• Glucose radiolabeled at specific Carbons
– Can determine fate of individual carbons
• Carbons 1,6 (Pyruvate Methyl)
– 1st cycle: 2,3 of oxaloacetate
– Starting at 3rd cycle ½ radioactivity  CO2/cycle
• Carbons 2,5 (Pyruvate Carbonyl)
– 1st cycle: 1,4 of oxaloacetate
– 2nd cycle: all eliminated as CO2
• Carbons 3,4 (Pyruvate Carboxyl)
– All eliminated at CO2 during Pyruvate  Acetyl-CoA
You are following the metabolism of
pyruvate in which the methyl-carbon is
radioactive: *CH3COCOOH.
-assuming all the pyruvate enters the TCA
cycle as Acetyl-CoA, indicate the labeling
pattern and its distribution in oxaloacetate
first formed by operation of the TCA cycle.
Amphibolic
Nature of
Citric Acid
Cycle
Generation of Citric Acid Cycle
Intermediates
Pyruvate Carboxylase
ATP
COOH
ADP + Pi
O
H3C
C
CH2
COOH
Pyruvate
+ HCO3–
(CO2)
O
Pyruvate
Carboxylase
C
COOH
Oxaloacetate
Animals and Some Bacteria
In Mitochondrial Matrix
Biotin Cofactor
(CO2 Carrier)
O
HN
H2 C
C
S
NH
O
CH
(CH2)4 C
C O
NH (CH2)4 CH
NH
Biotin
Lysine
Reaction Mechanism I
(Dehydration/Activation of HCO3–)
O
AMP
O
O
O P
O P
O–
O–
–
O
C
OH
O–
ATP
HN
O
HCO3
H2C
–
C
S
NH
CH
Biotinyl-Enzyme
O
(CH2)4 C
NH (CH2)4 Enzyme
ADP + Pi
O
O
–
O
C
N
H2 C
C
S
NH
CH
Carboxybiotinyl-Enzyme
O
(CH2)4 C
NH (CH 2) 4 Enzmye
Reaction Mechanism II
(Transfer of CO2 to Pyruvate)
O
–
O
O
C
C
CH 2
O
–
O
–
O
Pyr uvate Enolate
O
–
O
O
C
C
–
C
C
N
H 2C
CH 2
S
NH
Car boxybiotinyl-Enzyme
O
CH
(CH2) 4 C
Biotinyl-Enzyme
O
–
O
O
C
C
CH 2 C
O
O
–
Oxaloacetate
NH (CH 2)4
Enzyme
Fates of Oxaloacetate
Gluconeogenesis
ATP ADP + Pi COO–
COO–
C O +
CH3
Pyruvate
HCO3–
C O
Pyruvate
Carboxylase
CH2
COO–
Oxaloacetate
Regulation!
Citric Acid
Cycle
Regulation of Pyruvate Carboxylase
Allosteric Activator
Acetyl-SCoA
Glyoxylate Cycle
Plants and Some Microorganisms
In Glyoxysome
Net Reaction of Glyoxylate Cycle
2 Acetyl-CoA  1 Oxaloacetate
Net increase of one 4-carbon unit!
Net conversion of acetyl-CoA to glucose
Glyoxylate Cycle Enzymes
H2 C COOH
H2 C COOH
HC COOH
HO
Isocitrate
Lyase
CH COOH
Isocitrate
H2 C COOH
Succinate
CHO
+
COOH
Glyoxylate
CoA-SH
CHO
COOH
Glyoxylate
+
HO
O
H3C
C
S–CoA
Acetyl–SCoA
Malate
Synthase
CH COOH
H2C
COOH
Malate
Plants and Some Microorganisms
Glyoxylate Cycle and the Glyoxysome
Regulation of the Citric Acid Cycle
Regulatory Mechanisms
• Availability of substrates
– Acetyl-CoA
– Oxaloacetate
• Product Inhibition
– NADH
• Energy Charge
• Need for citric acid cycle intermediates
as biosynthetic precursors
• Demand for ATP
Pyruvate Dehydrogenase
Reaction and Regulation
NAD+
NADH + H+
O
CH3
C
O
COOH
+ CoenzymeA
SH
Pyruvate
CH3
PDH
(active)
NADH
Acetyl-CoA
PDH
Kinase
Pyruvate
ADP
Ca2+
C
S
CoA + CO2
Acetyl–SCoA
Insulin
Ca2+
PDH
Phosphatase
PDH- P
(inactive)
Free Energy Changes of Citric
Acid Cycle Enzymes
Table 17-2
Regulation of Citrate Synthase
• Pacemaker Enzyme (rate-limiting step)
• Rate depends on
• Availability of substrates
– Acetyl-SCoA
– Oxaloacetate
• Reduction Potential (NAD+/NADH)
• Energy Charge (ATP/ADP/AMP)?
• Inhibited by:
– Citrate
– Succinyl-CoA
Regulation of Isocitrate Dehydrogenase
• Allosteric Enzyme
– Energy Charge
• ATP - inhibitor
• ADP - activator
– Reduction Potential
• NADH - inhibitor
• NAD+ - activator
– Ca2+
Regulation of
-Ketoglutarate Dehydrogenase
• Inhibitors
– NADH
– Succinyl-SCoA
• Activator: Ca2+
Regulation of the Citric Acid Cycle
Figure 17-16
Shortened MCAT passage:
The citric acid cycle starts with a two carbon acetyl-group
from acetyl-CoA attaching to a four-carbon oxaloacetate
molecule to produce a six-carbon citrate. During
oxidative decarboxylation reactions, two carbons are lost
as CO2.
The citric acid cycle will result in a net gain of how many
carbons?
A. 0 carbons
B. 1 carbon
C. 2 carbons
D. 4 carbons
Which are the energy capture steps in the
CAC?
8
A.
B.
C.
D.
E.
1, 3 and 4
1 and 5
3, 4, 6 and 8
5, 6 and 8
3, 4, 5, 6 and 8
1
7
2
6
3
5
4
If acetyl-CoA labeled with 14C, as shown in the figure to the
right, were used as the substrate for the citric acid cycle,
which of the following intermediates would be produced
during the first round of the cycle?
OH H
O2*C
C C
CH2 CO2
H CO2
A)
OH H
O2C
C C *CH2 CO2
H CO2
B)
OH H
O2C
C)
C C
CH2 *CO2
H CO2
OH H
O2C *C C CH2 CO2
D)
H CO2
OH H
O2C C *C CH2 CO2
E)
H CO2
Germinating plant seeds can convert acetyl-CoA (obtained
from fatty acids stored as oils) into carbohydrates,
whereas animals are incapable of converting fatty acids
into glucose. This difference is due to the fact that:
A. animals have glycogen and don’t need to make glucose from
fatty acids.
B. plants use the glyoxylate cycle to convert two acetyl CoA to
oxaloacetate, a precursor for gluconeogenesis.
C. plant seeds use photosynthesis to make sugar.
D. animals use the citric acid cycle selectively for energy
production, whereas plants primarily use glycolysis.
E. B and D
• For each step of the TCA cycle:
– What do we get out of this?
– Thermodynamics?
– Is it Regulated? How?
• Where do the carbons of acetyl-CoA end up in
the TCA cycle?
• Why is the TCA cycle amphibolic?
• How are TCA cycle intermediates replenished?