Download CHAPTER 16 - CITRIC ACID CYCLE Introduction:

CHAPTER 16 - CITRIC ACID CYCLE
Introduction:
- Glycolysis and other catabolic pathways are oxidative. Electrons are stripped from a fuel
substrate (e.g., glucose) and passed to the appropriate electron acceptor, which thus is reduced.
Recall that in glycolysis, pyruvate was reduced to lactate (which is the same oxidation state as glucose)
under anaerobic conditions because need to rapidly regenerate NAD+, which had received electrons as
glyceraldehyde 3-phosphate was oxidized to 1,3-bisphosphoglycerate.
- Normally, in our cells, and in other aerobic organisms, electrons are passed to O2 as fuel
molecules are oxidized. When we are not exercising rigorously, pyruvate, the end product of
glycolysis, passes into the mitochondria, where it is oxidatively decarboxylated to acetyl CoA which
then enters the citric acid. As we will see in chapter 17, the reduced cofactors NADH and FADH2
then pass their electrons through a membrane-bound electron transport chain to the ultimate electron
acceptor, O2. Such oxygen-requiring processes are termed (cellular) respiration.
- Several other
fuels, including proteins (6 amino acids) and fats funnel metabolites into the citric acid cycle, just as
glycolysis funnels in pyruvate.
- The citric acid cycle also functions to provide reactants for a variety of biosynthetic pathways.
- Historically, the citric acid puzzle was solved in the ‘30's, although the final picture didn’t
come together until the early ‘50's when acetyl CoA was shown to be the catabolic intermediate that
brings pyruvate into the cycle.
- As glucose is broken down into smaller products, it becomes increasingly difficult to break
bonds. Acetyl CoA is essentially a 2-carbon fragment of glucose and cannot undergo either alpha or
beta cleavages. The citric acid cycle is a catalytic pathway whose purpose is to facilitate the
breakdown of acetate into CO2 . Acetyl CoA condenses with a catalyst (oxaloacetate), thus producing
a larger molecule (citrate) which can then undergo structural rearrangements that result in first a beta,
then an alpha cleavage. The catalytic process is cyclic because of the need to regenerate the catalyst.
Pyruvate
Acetyl CoA
Citrate
catalyst
beta
CO2
Regenerate
catalyst
alpha
CO2
- The catalytic nature of the citric acid cycle was originally suggested by the finding that addition
of small amounts of various dicarboxylic and tricarboxylic acids greatly accelerated the rate of oxygen
consumption in muscle tissue, beyond expectations based on the oxidative capacity of the acid itself.
Thus, these substances were catalyzing the oxidation of other foodstuffs.
- There are several oxidations which occur during the pyruvate 6 CO2 conversion in
mitochondria. There are three requiring NADH as cofactor and 1 requiring FAD. As we’ll see in the
next chapter, electrons from the reduced versions of these cofactors (NADH and FADH2) feed their
electrons into the electron chain, ultimately to O2. The electron transport process is energy-yielding and
is coupled to ATP formation (oxidative phosphorylation). The majority of the 36 to 38 ATP extracted
per glucose under aerobic conditions is via oxidative phosphorylations (3 per NADH and 2 per
FADH2).
Synthesis of Acetyl CoA
- It is useful to compare the aerobic conversion of pyruvate to acetyl CoA to the anaerobic
conversion of pyruvate to
acetaldehyde in yeast:
O
O
C
C
yeast
H
CH3
mitochondria
S CoA
O
C
O C
CH3
CH3
- Both of these reactions involve alpha decarboxylations, hence require thiamine as a cofactor.
See Figure 14-20 for a depiction of the thiamine-catalyzed alpha decarboxylation.
- Formation of acetyl CoA is a much more complicated process than the formation of
acetaldehyde and in eucaryotes is catalyzed by a multi-million molecular weight complex, the pyruvate
dehydrogenase complex, consisting of multiple copies of three enzymes, E1, E2 and E3 (E1 = pyruvate
dehydrogenase; E2 = dihydrolipoyl transacetylase; E3 = dihydrolipoyl dehydrogenase).
- E2 is the core of the complex and covalently binds lipoate via an amide linkage to a lysine
residue:
HS
HS
NH CH2
CH2 CH2
CH2 CH2
CH2 CH2 C
E2
O
Lipoate
Lysine
- The methylene groups of the lipoate together with the lysine R group forms a long “tether”
group that swings the sulfur groups, to which substrate binds, from E1 to E2 to E3.
R
1
N
C H
S
R
2
R3
O
+
O
C
Mech
C O
anism:
CH3
Let’s
pick
E1
up the
R
2
R
action
R O
O
1
C
N
C C OH
S
CH3
3
with
E1,
which
O C O
R
1
N
C
S
R
2
R
H
bound
CH3
thiamin
e:
H+
CH3
R3
C OH
3
C
R
2
sa
O
+
R
1
N
C H
S
contain
+
+ H+
R
1
N
R
2
C
S
R
3
(yeast only)
S
C OH
S
E2
Tether -
CH3
(pyruvate dehydrogenase only
R
2
R
3
HS
R
1
N
S
C
S
C OH
CH3
E2
Tether -
HS
R
1
N
R
2
S
C
Tether -
C OH
S
R
3
E2
CH3
B
+ H+
R
2
R
3
R
1
N
CH
S
HS
E2
S
+
C O
Tether
CH3
- At this point the pyruvate has undergone oxidative decarboxylation. A thioester linkage has
been formed between substrate and lipoate. An internal transfer of electrons has occurred such that the
original C2 of pyruvate has been oxidized to the oxidation state of a carboxylate group (S “owns” the
electrons in the C-S bond) and the lipoate has been partially reduced.
- Note that the lipoate group is capable of existing in either a completely reduced form or
oxidized form:
HS
HS
E2
Tether -
-H + H
+
+ H + H+
S
S
E2
Tether -
- E2 must now catalyze the transfer of the acetyl moiety from the lipoate to a CoA(SH)
molecule and the reduced lipoate must transfer a pair of electrons to an external electron acceptor
(NAD+):
HS
E2
S
C O
Tether
CH3
CoASH
HS
E2
HS
SCoA
C O
Tether
CH3
FAD (E2 - bound)
S
E2
S
FADH2
Tether
FAD
This reaction can be
summarized as follows:
CoA
Acetyl CoA
Pyruvate
Lipoate
Lipoate
(oxidized) (reduced)
FADH2 FAD
NAD
+
NADH
NAD+
NADH
O
C
S CoA
CH3
CO2
CH2
1
NADH
+
NAD
CO2
HO CCO2
CH2
CO2
C O
C O2
CH2
CO2
CH2
9
2
HC CO2
HO CH
CO2
NAD+
C O2
HO CH
NADH
3
CH2
CO2
CO2
CH2
HCCO2
O C
8
CO2
C O2
CH
HC
CO2
CH2
C O2
7
CH2
CO2
FADH2
6
CH2
CO2
O C
C O2
CH2
FAD
4
CH2
CH2
GTP
O C
GDP
S CoA
CO2
5
NAD+
NADH
Notes:
1. Acetyl CoA condenses with the catalyst, oxaloacetate, to enter the citric acid cycle. Citrate, the
product, has 6 carbons which can now be arranged to facilitate decarboxylations by either alpha or
beta cleavages.
2. Citrate is isomerized to isocitrate because the hydroxyl group on citrate is on a tertiary carbon and
cannot be oxidized to a carbonyl. Recall that the OH group on isocitrate is now on the portion of the
molecule originally derived from oxaloacetate. See Figure 20-15 to refresh your memory as to why the
OH never gets bound to a carbon originally from acetyl CoA.
3. Isocitrate is oxidized to oxalosuccinate, an unstable intermediate which spontaneously undergoes
beta decarboxylation to form alpha ketoglutarate (step 4.).
5. Alpha keto glutarate undergoes alpha decarboxylation to form succinyl CoA in a manner uncannily
similar to that of the pyruvate to acetyl CoA reaction considered in detail above.
6. Whereas the high-energy acetyl CoA was put to use to drive the otherwise energetically unfavorable
formation of citrate, the high-energy succinyl CoA is hydrolyzed to drive the formation of GTP, our
third (and last) substrate level phosphorylation.
7. - 9. The last three reactions regenerate the catalyst by successively forming a double bond, adding
water across the double bond and oxidizing the hydroxyl group to a carbonyl, yielding oxaloacetate.
Other notes:
- Because the citric acid cycle is an important source of precursors for other pathways (citrate,
oxaloacetate, succinyl CoA and alpha keto glutarate can be drawn off - see Figure 20-17), the
concentrations of citric acid cycle intermediates can fall to unacceptably low levels. Recall that because
of the catalytic nature of the cycle, these levels are fairly low to begin with.
To prevent this from happening, pyruvate can be converted to oxaloacetate (anaplerotic reaction). We
recognize this as the first step in
gluconeogenesis:
C O2
CO2
C O2
biotin
C O
CH3
C O
CH2
ATP
ADP
CO2
Amphibolic Nature of cycle
- In addition to catalyzing the conversion of acetyl CoA into CO2, the citric acid cycle also
ser
ves
Pyruvate
Fatty
acids
to
Cholesterol
pro
vid
e
Amino
Acids
Acetyl CoA
Citrate
Oxaloacetate
inte
Isocitrate
rme
diat
Malate
es
Alpha ketoglutarate
for
sev
Amino
Acids
Fumarate
Succinyl CoA
eral Amino
oth
Acids
er
pathways.
Succinate
Odd- chain
fatty acids
Amino
Porphyrins
Acids
- Because of the catalytic nature of the cycle, its intermediates are present in low, catalytic
amounts. Should the drain of these intermediates into any of these pathways, citric acid cycle activity
could be compromised, which is unacceptable. The following anaplerotic (“filling up”) reaction is one
of a few that replenish citric acid cycle intermediates:
O
O
O
C
O
C
C O
C O
CH2
CH3
Pyruvate
ATP ADP
C
O
O
Oxaloacetate
- You will recognize this reaction as the first step in gluconeogenesis. Be sure to make note of
this in your metabolic road map.
Regulation
- The four NADH, 1 FADH2 and 1 GTP obtained from converting pyruvate to CO2 results in 4
x 3 + 1 x 2 + 1 = 15 ATP per pyruvate, or 30 per glucose. Such a large amount of ATP necessitates
tight regulation of pyruvate to acetyl CoA as well as the citric acid cycle itself.
- Regulation of the pyruvate dehydrogenase complex occurs mechanistically by product
inhibition and covalent modification. NADH and acetyl CoA as products thus compete for binding
sites on E2 and E3. Since NADH is also an electron donor, large amounts of NADH (or large
[NADH][/NAD+] ratios) can reverse the flow of electrons, ultimately preventing the oxidative
decarboxylation of pyruvate by E1.
- E1 is also subject to covalent modification by a regulatory kinase (which is allosterically
activated by the high-energy indicator, ATP). Phosphorylation of E1 results in its inactivation. Note
that insulin, which we have previously seen to have antagonistic effects with respect to glucagon and
epinephrine, and which terminates glycogen breakdown and initiates glycogen synthesis, reverses the
inactivation of E1 by activating the phosphoprotein phosphatase which removes the phosphate group on
E1. This is consistent with insulin’s role in accelerating glucose uptake as well as stimulating cellular
processes that use up or store glucose.
- Regulation of the cycle itself occurs at the three exergonic reactions, i.e., the synthesis of
citrate (involving the hydrolysis of the high-energy thioester bond in acetyl CoA) and the two
decarboxylations. Although allosteric modulators are involved in regulation of the cycle (as high-energy
indicators, they have an inhibitory effect), the primary mechanisms are substrate availability(1),
product inhibition(2) and competitive feedback(3).
Pyruvate
NADH (2)
*
Acetyl CoA(1)
NADH (2)
*
Citrate
Oxaloacetate(1)
Isocitrate
* NADH (2)
Malate
Alpha ketoglutarate
* NADH (2)
Fumarate
Succinyl CoA
(3)
Succinate
- Note that the (3) next to succinyl CoA implies that succinyl CoA inhibits via competitive
feedback inhibition. In this case the reaction inhibited in the citrate synthase reaction, which is also
inhibited by acetyl CoA. See Figure 16-14, p. 486.
Glyoxalate Cycle - Seeds store fats as both a source of energy and precursors for carbohydrate. The
glyoxalate cycle is properly thought of as a variation of the citric acid cycle.
O
C
S CoA
CH3
CO2
CH2
NADH
+
NAD
HO CCO2
CH2
CO2
C O
C O2
CH2
CO2
CH2
HC CO2
HO CH
CO2
CO2
C O2
O
HO CH
CH2
C
CO2
CH3
NAD+
NADH
S CoA
H
O
CO2
1
CH2
C
2
HCCO2
O C
C O2
CO2
C O2
CH
HC
CO2
CH2
C O2
CH2
CO2
FADH2
FAD
CH2
C O2
CH2
CH2
CO2
O C
CO2
CH2
GTP
O C
GDP
S CoA
NAD+
NADH
- The glyoxalate cycle includes citric acid cycle reactions up to the formation of isocitrate.
Isocitrate is then cleaved to form glyoxalate and succinate in the first reaction unique to seeds (reaction
#1). Then glyoxalate is added to a second acetyl CoA in reaction #2 to form citrate.
- In this way, via the actions of two additional enzymes, isocitrate lyase and malate synthase,
seeds avoid the decarboxylation steps of the citric acid cycle. The catalyst can be regenerated from
succinate, one product of the lyase reaction, so the malate synthesized from the two acetyl CoA can be
converted to oxaloacetate, then glucose via gluconeogenesis. See Figure to see how the reactions are
compartmentalized between the various organelles.