Chapter 16 Citric Acid Cycle
Now will get the rest of the E out of pyruvate by completely oxidizing to CO2 and H2O
Aerobic phase called respiration
More narrow definition, cellular respiration to differentiate from organismal breathing
Actually part of a 3 step process (figure 16-1)
Step 1 last chapter
Step 2 chemical conversion of pyruvate to CO2 but H transferred to carrier NADH
and FADH2 doesn’t generate much E, only a single GTP
Step 3 NADH and FADH transfer H to O to make H2O. Here is where we get the
bulk of the E, and lots of ATP via oxidative phosphorylation. Chapter 19
Since early conditions on earth didn’t have O2 this is thought to have evolved later
Main bulk of reaction will look at called the citric acid cycle or the Kreb’s cycle after its
discoverer.
Several places to feed other compounds into this cycle, and several places to siphon off
product for synthesis of other things, so has a central role in metabolism, and has
complex control
(you thought glycolysis was tough)
16.1 Production of acetate
Several different ways compounds can enter the citric acid cycle. Many AA,
pyruvate from glycolysis, and fatty acids enter as an acetate groups attached to
coenzyme A
While this is performed as a single enzymatic reaction, is very complex multi
enzyme complex called pyruvate dehydrogenase complex (PDH) and contains
5 different coenzymes derived from vitamins and needs a bit of an explanation
same complex structure is also used by two other enzymes system, so if you get
this down you will understand the others
Also note the location. We have now shifted to an enzyme complex in the
mitochondria. Glycolysis was done by complexes in the cytosol, and at this point
the pyruvate needs to move into the mitochondria, and all subsequent reaction
we will talk about take place in the mitochondria
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A. Oxidization of pyruvate to acetyl-CoA and CO2
overall reaction takes place in pyruvate dehydrogenase complex
reaction called oxidative decarboxylation
ÄG = -33.4 kJ/mole
Very large and irreversible
note in this reaction 2 electrons and an H are given to NAD+ as a hydride
(H-) in the mitochondria the NADH. This NADH is then used to generate
about 2.5 ATP through oxidative phopshorylation (chapter 19)
B. Pyruvate Dehydrogense Complex - 5 coenzymes
the above reaction involved 3 different enzymes and 5 different
coenzymes (NAD, FAD, TPP, Lipoate and coenzyme A
(Just look at structures now, will talk about mech next page)
NAD (nicotinamide adenine dinucleotide) derived from vitamin niacin or
nicotinic acid. An electron carrier that works in 2 electron steps. Had in
chapter 14 skip
FAD (flavin adenine dinucleotide) derived from riboflavin (Vitamin B2),
another electron carrier, but can work in 1 electron steps Structure 13-27
Look at here, but will give more details in a minute
TPP (thiamine pyrophosphate) derved from thiamine (vitamins B1) used to
manipulate chemistry adjacent to carbonyl groups Structure and typical
mech figure 14-15 (look at details now)
CoA (coenzyme A) derived from pantothenic acid, used as a soluble
acetate group carrier that can travel from one enzyme to the next in
solution. Acyl group attached to Co A is high energy intermediate and is
readily donated to other groups Structure 16-3 (look at details now)
Lipoic acid (can be synthesized in you body so not a vitamin) used as an
acetate carrier that is anchor in a complex undergoes reversible thiol
oxidation and reduction, much like cystine in a protein. Structure 16-4
(Look at now)
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C. Complex contains 3 enzymes
pyruvate dehydrogenase (E1)
Dihydrolipoyl transacetylase (E2)
Dihydrolipoyl dehydrogenase (E3)
Many copies of each, exact composition varies in each organism
Bovine kidney structure (Figure 16-5)
About 50 nm diameter. 5x larger than entire ribosome!
Core cluster 60 copies of E2
3 functional domains
Lipoyl domain - contans 2 lipoic Acids
E1-E3 binding domain
Acyl transferase domain
Domains separate linker of 20-30 rich in Ala, Pro,+/to keep domains separate
E1
Each with a TPP
E3
Each with a bound FAD
Also two regulatory proteins
A protein kinase
A phosphoprotein phosphatase
Basic structure conserved during evoluion
D. Reaction Mech involves substrate channeling
overall reaction mech shown in figure 16-6
step 1 pyruvate bound to TPP on E1 and is decarboxylated
See TPP reaction mech figure 14-15 for more details
step 2 the Lipoic acid (oxidized) on E2 get reduced and removes the
acetate product to regenerate E1
This lipoic acid is attached to a lysine (figure 16-4)
This makes it a long attachment that can move
Note: this motion is shown in figure
step 3 free CoA comes in, and acetate transferred to CoA which can leave
step 4 lipoic acid still reduced, so transfers electron to E3 bound FAD so it
is oxidized agin and FAD reduced to FADH2
step 5 NAD+ comes in and takes electron from FADH2 so enzyme is
regenerated
NADH is released to find its fate in the mitochondria
Note how this reaction mechanism is really streamlined by use of lipoic
acid to move things around. Substrates don’t have to diffuse one and off,
they are there ready to react. An example of substrate channeling
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Mutations in any protein, or vitamin deficiencies can really screw things up
Beri-Beri disease results from deficiency in Thiamine, characterized by
loss of neural functions
-originally seen in populations living on polished rice (polishing the
rice removed the thiamine)
- fish project linking lack of thiamine to fish reproduction in lake
Oahe
Can also be seen in alcoholics because getting calories without
vitamins
16.x FMN & FAD(actually chapter 13 535 & 536)
FMN Flavin mononucleotide
FAD Flavin adeninedinucleotide
Proteins that use either of the F’s are called flavoproteins
Figure 13-27 page 536
another electron carrier
another vitamin - riboflavin (B2)
There doesn’t seem to be much information for disease state due to lack
of riboflavin
Differences from NAD(P)
1. Usually very tightly bound to enzyme so doe NOT diffuse around cell
(Sometimes covalently attached)
2. As shown in figure can take one or two H+ + eSo seen in a wider range of reactions
To differentiate between state
FMN, FMNH@, FMNH2
FAD, FADH@, FADH2
Also have shift in absorbance Oxidized max about 570, reduced max around 450
Since tightly bound to protein, protein has great influence of Eo. So it varies from
protein to protein.
Flavoproteins tend to be very complex, often other thing involved in e transfers
including other F’s, inorganic ions (often Fe or Mo)
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16.2 The Citric Acid Cycle
first example of a cycle
Figure 16-7
start by adding acetate from acetyl CoA to 4C oxaloacetate to get citric acid
(Hence name of cycle)
Then thru series of steps 2 CO2 are removed (NOT the C rom the acetate!)
And 3 NADH, 1 FADH2 and 1 GTP generated and oxalacetate regenerated to
begin cycle again
Cycle is nice 1 oxaloacetate can oxidized infinite # of acetate
4, 5, & 6C intermediates serve both and sources for synthesis of other
compounds, and a ways to bring in other compound for oxidation
In eukaryote entire cycle and the subsequent oxidative phosphorylation take
place in mitochondria in prokaryote take place in cytosol with oxidative
phosphorylation taking place in plasma membrane
A. Chemical Sense of the Citric Acid Cycle
First of all the Acetyl CoA that we are oxidizing in the citric Acid cycle does not
come only from pyruvate, but it can come for fatty acid oxidation or other
carbohydrates or proteins, so we have many oxidative pathways focused down
to this cycle Figure 15-1
Second of all, since we have lots of different pathways focused on this one
method of oxidation, we want it to be as efficient as possible so we get the most
energy we can out of the acetate group.
If oxidize the acetate group in the simplest manner we simply complete the
oxidation of the COOH group and go to methane (CH4) and CO2. The problem
with this is that methane is very stable, and only a few methanotropic bacteria
have the enzymes needed to break it down. So this would throw away lots of
our potential energy. Instead we need to work on that CH3 group and get as
much of its energy as we can.
Back in chapter 13 we talked a bit about the chemical logic, and one of the thing
we saw was that methyl groups are more reactive when the are directly attached
to a COO group or 1 methyl group away from a COO group. Thus what the Citric
Acid cycle is going to to is to take that CH3 group and put it adjacent to other
groups to it becomes more reactive, and we will use step by step reactions to
slowing oxidize it down by small steps to get the most energy was can out of it.
Watch as we do different reactions to put different functional groups beside that
CH3 so we can break its bonds efficiently
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B. Citric Acid Cycle (8 steps)
1. Formation of citrate
Citrate synthase
ÄG0' = -32.2 kJ/mol
citroyl-CoA is seen as a transient intermediate in the enzyme’s
active site’
See Mech figure 16-9
[oxaloacetate] is normally very low
thus, if this reaction didn’t have such a large, favorable ÄG might
go in reverse
So large favorable ÄG necessary start to the cycle
Co-A liberated and can go back and get another acetate
is an ordered reaction (oxaloacetate must bind first then acetyl
CoA)
--doesn’t have a binding site for CoA until oxaloacetate binds and
makes induced fit to open up CoA binding site
– after citronylCoA formed in binding site a second induced fit to
bring in to split CoA off the citrate
these two conformational event use to keep enzyme from simply
binding CoA and releasing the acetate group in a useless manner
See Figure 16-8
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2. Formation of Isocitrate via cis-Aconitate
Aconitase or aconitate hydratase
ÄG0' = 13.3 kJ
reversible
intermediate usually does not come off the enzyme
At pH 7.4 and 25oC, equilib is 90% citric, 10% isocitrate
but in cycle isocitrate is rapidly used up so []9 so reaction pulled to
right
aconitase contains and iron-sulfur cluster (figure 16-10) that acts
both in catalysis and for binding
Box 16-1 interesting aconitase is also involved in iron regulation
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3. Oxidation of Isocitrate to á-Ketoglutarate and CO2
Isocitrate dehydrogenase
Mech figure 16-11
ÄG0' = -20.9 kJ/mol
in eukaryotic cells 2 isozymes of this enzyme
Mitochondrial form used NAD+
cytosol form uses NADP+
cytosol form thought to be used to make NADPH for synthetic
pathways
Note the CO2 is NOT a C from the acetate
4. Oxidation of á-ketoglutarate to Succinyl-CoA and CO2
á-ketoglutarate dehydrogenase complex
ÄG0' = -33.5 kJ/mol
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virtually identical to pyruvate dehydrogenase complex in both
structure and function
E1 enzymes are similar, but not identical, need to binds different
substrates
E2 very similar
E3 virtually identical
probably share common evolutionary origin
5. Conversion of Succinyl-CoA to succinate
S
Succinyl-CoA synthase (succinate thiokinase)
ÄG0' = -2.6 kJ/mol
Mechanism figure 16-13
Pi is used to displace CoA from succinate
the phosphoanhydride then phophoylates a His in the active site to
generate suc
Suc floats off, and the phosphoenzyme then can add phosphate to
ADP or GDP
enzyme is 2 units
á 32,000 binds the CoA and gets phosphorylated at his 246
â 42,000 binds ADP or different â GDP
active site at interface
substrate level (direct phosphorylation)
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If make GTP can use nucleoside diphosphate kinase to transfer to
ADP for no cost
Note: above mechanism is almost exactly the same as one used in
pyruvate dehydrogenase and the oxidation of isoleucine, leucine or
valine! Figure 16-12
6. Oxidation of Succinate to Fumarate
Succinate dehydrogenase
ÄGo’ =0
This enzyme tightly bound to inner mitochondrial membrane
Only membrane bound enzyme in the whole cycle
three different iron sulfur clusters and 1 covalently bound FAD
This FAD passes electrons to membrane bound portion of oxidative
phosphorylation
Will get about 1.5 ATP for this FADH2
Malonate (not malate) very similar to Succinate (See margin page
647 for structures)
acts as competitive inhibitor of this enzyme, and therefor for entire
TCA cycle
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7. Hydration of fumarate to malate
(Trans)
Fumarase (or fumarate hydratase)
ÄG0' =-3.8 kJ/mole
highly stereospecific
trans not cis double bond
make L not D
(Will do in lab 2nd semester)
8. Oxidation of Malate to Oxaloacetate
L-malate dehydrogenase
ÄG0' = 29.7 kJ/mol
very unfavorable under standard state conditions
but conc of oxaloacetate in cell extremely low (<10-6) due to initial
reaction of TCA cycle so reaction keeps going
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C. Energy in the cycle is efficiently conserved
Total for 1 turn of the cycle
CH3CO-SCoA in
2 CO2 Out
3 NADH out
1 FADH2 out
1 GTP or ATP out
The 2 C that appear as CO2 are NOT the same that came in the
acetate
Will see in chapter 19 that NADH yields about 2.5 ATP and FADH2
yields about 1.5 ATP
So TCA cycle give 3(2.5) + 1(1.5) + 1 ATP
= 10 ATP
Lets’ put the whole total together
Chapter 15
1 glucose to 2 pyruvate + 2 ATP + 2 NADH
(~2.5 ATP/NADH = 5 ATP)
= 2 + 5 = 7 ATP
2 Pyruvate to acetyl Co A yield 2 NADH for another 5 ATP
And 2 acetyl-CoA thru the TCA give us 20 ATP
Thus our total is
Glucose 6 6 CO2 + 7+5+20 ATP = 32 ATP
32 x 30.5 kJ/mole = 976 kJ of E conserved
Glucose to CO2 could yield 2840 so
976/2840 = 33.3% efficient
D. Why is oxidation of acetate so complicated?
8 step cycle to oxidize 2 C seems a bit excessive
Nature usually much more economical
Job is not simple oxidation but to be hub of metabolism
Can use as source or drain of 4, 5, and 6C compounds
Also product of evolution, so may not be cleanly designed
Some modern anaerobes use incomplete cycle just to synthesize
various precursors
13
E. Citric Acid cycle components as intermediates
TCA is amphibolic pathway
Serves both anabolism and catabolism (build up and tear down)
á-ketoglutarate and oxaloacetate simple transamination from glu and asp
And other amino acid
oxaloacetate can also be converted to glucose in gluconeogenesis
Succinyl-CoA intermediate in synthesis of heme ring
see figure 16-16 for summary
F. Anaplerotic Reactions
if an TCA intermediate removed as a synthesis precursor, then TCA can’t
carry on because is a cycle
Need to replace intermediates when they are siphoned off
anapleroic reaction, one that replaces missing intermediates
Most common anaplerotic reaction shown in table 16-2 also in red on
figure 16-16
These reactions vary from one tissue to another
One of main reactions is carboxylation of pyruvate by pyruvate caboxylase
To make oxaloacetate
Take an ATP for E
Highly regulated
Saw this last chapter as way to pyruvate to oxaloacetate to PEP
Virtually inactive unless acetylCo-A present
(Indicating that not enough TCA is around to burn it up)
All of the anaplerotic reaction are going to be regulated
G. Biotin
the mention of pyruvatecarboxylase introduces another vitamins we can
look at Biotin
Specialized carrier of CO2
Structure/mech shown in figure 16-17
In this enzyme covalently attached to end amino group on a lys
Making another long flexible arm
In enzyme step 1 carboxylation of biotin
Step 2 transfer of carboxyl to pyruvate
Occur at separate sites to long flex is used to move C from
one site to the other
Biotin required in human diet, but synthesized by many
intestinal bacteria - so deficiency is rare
Mostly occurs when eat large amounts of raw eggs
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Avidin is an egg protein that binds biotin
Once bound can’t be absorbed by intestine
But if egg is cooked, protein denatured, can’t bind
16.3 Regulation of TCA cycle
flow from pyruvate to cycle has 2 control points
conversion of pyruvate to acetyl Co A
Pyruvate dehydrogenase (-33.4 kJ)
Entry of acetyl CoA into cycle
Citrate synthase (-32.3 kJ)
At first this seems surprising because you have 2 control points in a row
but control at other points important because acetyl CoA can have other
sources
Fatty acid oxidation
Some amino acid oxidation
and acetyl CoA can go other places
FA synthesis for one
A. Production of Acetyl CoA by Pyruvate Dehydrogenase
both allosteric and covalent modification types of control
Complex inhibited by ATP, acetyl-CoA, NADH ( reaction products)
Inhibition enhance by long-chain fatty acids
activated by AMP, CoA, and NAD+ which accumulate when too little is
introduced to the cycle
Net turned off when have lots of E, turned on when E is low
In vertebrates additional covalent control
Inhibited by reversible phosphorylation of a ser on E1 by a kinase
Kinase turned on by excess ATP
P is removed by a specific phophatase
Plant may also have phosphorylation control, but not entirely clear
E coli definitely not phosphorylation control (on top of allosteric)
B. TCA cycle is regulated at its three exergonic steps
citrate synthase (-32.2 kJ)
isocitrate dehydrogenase (-20.9 kJ)
á-ketoglutarate dehydrogenase (-33.5 kJ)
see figure 16-19
#1 9 by NADH, citrate, ATP 8ADP
#2 9 by ATP 8 Ca or ADP
#3 9 by succinyl CoA 8 Ca2+
In general well matched, so nothing accumulates
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C. Substrate Channeling through Multienzyme complexes
Originally thought that all enzyme in TCA pathway were simply freely
dissolved in the mitochondrial matrix
May have simply because that is how we isolated them
Growing evidence that many or all are part of multi-enzyme
complexes called metabolons
So not feely soluble enzymes, but highly organized with channeling
of intermediates to make more efficient
D. Some mutations in TCA cycle lead to Cancer
You can read details if interested
16.4 The Glyoxylate Cycle
The two steps PEP to pyruvate and pyruvate to acetyl CoA are strongly
exothermic, and essentially irreversible
Thus once C has gotten down to the pyruvate or acetyl-CoA level, it is committed
to oxidation can cannot be recycled back to the gluconeogenesis pathway to
make carbohydrates
Thus all vertebrates cannot convert fatty acids or acetate into carbohydrates.
Plants can do this because they have an additional cycle called the glyoxylate
cycle.
And this cycle is now discussed. I am going to show that I am a vertebrate
chauvinist and I am not going to be politically correct and give the plants their fair
time. Thus we will not study this cycle. But if you are a Plant person, this should
be required reading.