CITRIC ACID (KREB’S, TCA) CYCLE
Date:
Time:
Room:
Lecturer:
September 2, 2005 *
10:40 am- 11:30 am *
G-202 Biomolecular Building
Steve Chaney
515A Mary Ellen Jones Building
stephen_chaney@med.unc.edu
966-3286
*Please consult the online schedule for this course for the definitive date and time for this lecture.
Office Hours: by appointment
Assigned Reading: This syllabus.
Key Concepts vs Supplementary Information: Because this syllabus is meant
to replace the need for a Biochemistry textbook, it contains a mixture of
information that is critical for you to know and information that serves to
illustrate and explain the key points. I have attempted to emphasize important
terms, definitions and concepts in red, and have listed key points after each
section of the syllabus. Illustrative and supplementary information is indicated in
italics.
Overall Objectives for Carbohydrate Metabolism:
By the end of this course, you should:
1) understand how carbohydrate metabolism normally responds in the fed state,
the fasting state, and during exercise.
2) understand how carbohydrate metabolism is altered by diabetes and the
metabolic response to trauma and surgery.
3) understand the relationship between obesity, exercise, insulin resistance, and
diabetes.
4) understand basic priciples of diet composition and weight management.
Lecture Objectives: At the conclusion of this lecture you should:
1) know the vitamins and cofactors that are required by the citric acid cycle
(especially for the pyruvate dehydrogenase and α-ketoglutarate
dehydrogenase reactions).
2) know how the citric acid cycle is regulated.
3) know the role that the citric acid cycle plays in carbohydrate, amino acid, and
fat metabolism.
4) know the role that the citric acid cycle plays in the interconversion of
carbohydrates, amino acids, and fats.
Important concepts:
1.
All fuel molecules are oxidized to citric acid cycle intermediates.
2.
The citric acid cycle is important for the biosynthesis of glucose, lipids, and some
amino acids.
The citric acid cycle breaks down a 6 carbon compound (citrate) to a 4 carbon
compound (oxaloacetate). The high energy electrons are transferred to co-enzyme
carriers (NAD+ and FAD) and destined for oxidative phosphorylation. The carbons are
removed as CO2, and the H+ will follow with the electrons to O2 to form H2O. The
citric acid cycle enzymes are located in the mitochondria, but they are mostly soluble
enzymes located in the mitochondrial matrix.
A.
Pyruvate dehydrogenase complex (PDH) Pyruvate dehydrogenase is a large
enzyme complex in the mitochondrion consisting of 3 different types of enzyme subunits.
It is the enzyme that connects the glycolytic pathway to the citric acid cycle.
-
Connects Glycolysis to C.A.C.
CO2
C
CoASH
SCoA
NADH
O
CH3
C
NAD+
pyruvate
CO2
O
CH3
acetyl CoA
Note that 4 of the coenzymes used in this reaction are derived from substances
that must be obtained in the diet.
thiamine or vitamin B1 - thiamine pyrophosphate (TPP)
pantothenic acid - coenzyme A (CoASH)
riboflavin – FADH2
niacin - NADH
B. Overview of the Citric Acid Cycle
COOC
CH3
CoASH
NADH
CO2
SCoA
C
CO 2C
NADH
CO2CHOH
CH 2
pyruvate
O
pyruvate
dehydrogenase
O
CO 2-
CH3
acetyl CoA CoASH CH 2
O
CH 2
HO
Citrate
synthase
CO 2 -
C
CH 2
CO 2-
CO 2-
OAA
citric
acid
Malate
dehydrogenase
CO2
NADH
Succinyl CoA
synthetase
CO2-
malate
CO2
DH 2
FA
CH2
CH2
CO2-
-
CH2
CH2
CH2 CoASH
CO2succinate GTP
O
C
C
αKG
NADH
CH2
CO2-
CO2-
CO2
CoASH αKG
dehydrogenase
SCoA
O
succinyl CoA
The citric acid cycle contains a cyclic sequence of 8 enzymes that are so arranged
that they perform molecular rearrangements of intermediate metabolic compounds
to prepare them for decarboxylation and dehydrogenation.
.Selected
Enzymatic Steps
1.
Citrate synthase: this is a 2 step reaction: an aldol condensation of
oxaloacetate and acetyl CoA, followed by hydrolysis to yield citrate and free CoA. The
hydrolysis step is not easily reversible.
SCoA
C
CO 2C
O
CH 2
CO 2-
O
CO 2 -
CH3
acetyl CoA CoASH
Citrate
synthase
HO
CH 2
C
CO 2 -
CH 2
CO 2 -
citric
acid
2.
α-ketoglutarate dehydrogenase: a multi-enzyme complex very similar
to pyruvate dehydrogenase. This step is irreversible. It also produces NADH.
CO2CH 2
CO2-
CH 2
CH2
CH2
C
O
NADH
SCoA
CO2-
C
αKG
CO2
αKG
dehydrogenase
CoASH
O
succinyl CoA
3.
succinyl CoA synthetase: succinyl CoA has a high negative ΔG°' of
hydrolysis, and can, therefore, be coupled to the direct phosphorylation of GDP →
GTP (which is equivalent to ATP); this reaction is fairly reversible. This is an example
of substrate level phosphorylation.
Succinyl CoA
synthetase
CO2-
CO2-
CH2
CH2
CH2
CH2 CoASH
C
-
CO2
succinate GTP
4.
SCoA
O
succinyl CoA
malate dehydrogenase: this is a good example of a reaction that has a
net flow opposite to an unfavorable Keq. That is the oxidation of
malate by NAD+ to produce oxaloacetate + NADH + H+ has a ∆G°' of
+ 7 kcal/mole.
CO 2
C
NADH
CO2CHOH
CH2
O
CH 2
CO 2-
OAA
Malate
dehydrogenase
CO2-
malate
Question: What can you deduce from the above direction of this reaction about the
respective physiological concentrations of malate and oxaloacetate?
Net Energy Yield of the Citric Acid Cycle
For each acetyl group used up, the cycle produces:
3 NADH + 1 FADH2 + 1 GTP + 2 CO2
Since each NADH can result in 3 ATP, and each FADH2 can result in 2 ATP, the
net high energy production is 12 ATP. Therefore, the total energy from 1 glucose is:
1 glucose → 2 pyruvate: 2 ATP + 2 NADH (= 4 ATP)
2 pyruvate → 2 acetyl CoA: 2 NADH
2 acetyl CoA → 4 CO2 : 2 x 12 ATP
6 ATP
6 ATP
24 ATP
- 6 in cytosol
- 30 in
mitochondria
36 ATP
It should be self-evident why the 36 ATP that result from the complete
oxidation of glucose are far preferable to the 2 ATP that are derived by glycolysis
alone.
C.
Regulation of the Citric Acid Cycle
The citric acid cycle is regulated at multiple points. However, in general it is
safe to say that it is inhibited by ATP and NADH. The inhibition by NADH keeps it
tightly regulated by oxygen supply, since NADH is converted to NAD+ by oxidative
phosphorylation. The inhibition by ATP keeps the citric acid cycle in balance with
energy supply. When ATP (energy supply) is high, the citric acid cycle is inhibited and
precursors to the citric acid cycle (pyruvate, acetyl CoA and amino acids) are diverted
into other pathways.
Acetyl CoA, citrate, and succinylCoA are the end products of individual steps in
the citric acid cycle and their accumulation inhibits the step involved in their production.
That, of course, results in inhibition of the cycle as a whole. Finally, Ca++ stimulates
the citric acid cycle at several points. This is important because electrical stimulation of
the muscle causes an increase in intracellular calcium levels. Thus, during exercise
the citric acid cycle will be maximally stimulated in muscle. The regulation of the citric
acid cycle is summarized below.
Pyruvate
+
e-
Ca2+
Acetyl-CoA
-
Oxaloacetate
Citrate
Malate
Fumarate
Isocitrate
+ Ca2+
NADH
Succinate
e-
α-Ketoglutarate
GTP Succinyl-CoA
-
+
Ca2+
e-
ATP
Ox Phos
Regulation of the Citric Acid Cycle (animation available in PowerPoint)
D.
Relationship Between the Citric Acid Cycle and Other Metabolic Pathways
1.
Acetyl CoA is both the final product of fatty acid degradation and the
first building block for fatty acid synthesis.
2.
Several citric acid cycle intermediates can be converted to amino acids
by simple transamination reactions (see below). Thus, the citric acid cycle can be
involved in amino acid synthesis, degradation, or conversion to oxaloacetate for
gluconeogenesis.
αKG
+
Glu
Asp
+
OAA
CO2-
CO2-
CO2-
CO2-
CH2
CH2
CH2
CH2
+ CH
CH2
C
NH3+
CO2-
O
+
CH2
NH3+
CH
CO2-
C
O
CO2-
CO2-
α-Ketoglutarate Aspartate
Glutamate
C.A.C. and Other Pathways
Oxaloacetate
protein
Glucose
a.a.’s
PEP
protein
Alanine
fats
pyruvate
CO2
acetyl CoA
a.a.’s
Asp
OAA (C4)
fatty
acids
citrate (C6)
CO2
αKG (C5)
NADH + FADH2
Val, Ile
protein
a.a.’s
glutamate
succinate (C4)
ATP
protein
CO2
urea
cycle
porphyrins
hemoglobin
Interconversions of the Citric Acid Cycle (animation available in PowerPoint)
Thus, the citric acid cycle:
1.
is the final step in the conversion of all foods to CO2 and H2O.
2.
is the major source of reducing equivalents (NADH and FADH2) used
by the cell to generate ATP (via oxidative phosphorylation).
3.
is the central pathway that interconnects all others.
a.
Excess carbohydrate can be converted to protein* and fat.
b.
Excess protein can be converted to carbohydrate or fat.
c.
However, net conversion of fatty acids to carbohydrate and most
amino acids is not possible.
*Note: Some amino acids cannot be synthesized by the body and are, therefore,
essential components of the diet. In the absence of these essential amino acids,
net conversion of carbohydrate to complete proteins is impossible.
4.
The citric acid cycle requires a constant supply of oxaloacetate to keep
going.
(What enzyme supplies oxaloacetate when we break down carbohydrate?)
(Why is this enzyme activated by acetyl CoA?)
5.
Since oxaloacetate is depleted by gluconeogenesis, Dr. Atkins has postulated
that fat calories won’t be utilized in the absence of carbohydrate. Based on
what you now know about the citric acid cycle, what is the basic falacy of this
hypothesis? (Where does oxaloacetate come from when we break down
protein?)
Why Does Atkins Diet Appear to Work?
H2O retention (short term)
CHO
long term: wt loss = caloric balance
weight
high CHO
high fat
time
Any healthy diet will do
Main argument against Atkins
= unhealthy diet
Low carbohydrate versus low fat diets will be covered in more detail in lecture.
________________________________________________________________________
Key Points about the Citric Acid Cycle
1.
Cofactors for the pyruvate dehydrogenase reaction and their corresponding
vitamins.
thiamine pyrophosphate
lipoic acid
coenzyme A
FADH2
NAD
2.
thiamin
pantothenic acid
riboflavin
niacin
Regulation of C.A.C.
When O2 limiting it causes an increase in [NADH] which causes inhibition
(NADH = direct end product C.A.C.).
When cell has more energy than it needs the concentration of [ATP]
increases, which causes inhibition.
ATP = end product C.A.C. and ox. phos.
This diverts pyruvate, acetyl CoA and amino acids to other pathways
3.
C.A.C. and Other Pathways
It is the final step for conversion of all foods to CO2 and H2O.
It is the central pathway that interconnects all others.
CHO can be converted to protein or fat.
Some protein can be converted to CHO or fat.
Fat cannot be converted to CHO or protein.
OAA is needed to keep the C.A.C. going.
________________________________________________________________________
SAMPLE QUESTIONS
Each of the following questions has one correct answer.
1.
The overall reaction of the pyruvate dehydrogenase complex produces: NADH,
H+, CO2, and
a.
b.
c.
d.
e.
2.
lactate
oxaloacetate
citrate
ATP
Acetyl CoA
The citric acid cycle and oxidative phosphorylation both occur in the:
a.
b.
c.
d.
e.
lysosomes
nucleus
zymogen granules
cytoplasm
mitochondrion
3.
The citric acid cycle "begins" with citrate synthase catalyzing the formation of
citrate from:
a.
b.
c.
d.
e.
succinyl CoA + pyruvate
acetyl CoA + pyruvate
acetyl CoA + oxaloacetate
furmarate + oxaloacetate
pyruvate + lactate
Answers
1.
2.
3.
e
e
c
Comments
1.
Pyruvate dehydrogenase is a key enzyme. You are, therefore, asked to remember
that it converts pyruvate to acetylCoA.
2.
You need to know the location of metabolic pathways. e is correct.
3.
Again this is a key enzyme. You are asked to remember the substrates.