CITRIC ACID CYCLE (Kreb’s cycle/TCA cycle)
The citric acid cycle is the final common pathway for oxidation of carbohydrates, lipids and many amino acids. It takes place in aerobic organisms within cells that have mitochondria. Its main purpose is to oxidize acetyl-CoA and concomitantly reduce NAD+ and FAD. Re-oxidation of the reduced co-enzymes in the mitochondrial respiratory chain uses molecular O2 and generates ATP. The cycle also provides biosynthetic intermediates for other metabolic pathways.
4
GT P/AT P
LIPID CARBOHYDRATE
PROTEIN
Acetyl -CoA C2
O2 C
NADH ETS
C4
2 C6 C4
C C4
ETS NADH
FADH2 ETS
C4
CYCLE CITRIC ACID
C C5
O
NADH ETS
C6
Fig 5.3.1. Overview of the citric acid cycle (ETS = Electron Transport System)
REACTIONS OF THE TCA CYCLE
Each turn of the TCA cycle is constituted by 8 reactions, catalyzed by the sequential action of 7 enzymes and one multi-enzyme complex. The enzymes are present in the mitochondrial matrix, in close proximity to the electron transport chain on the mitochondrial cristae. The essential precursors for the TCA cycle are acetyl-CoA and oxaloactetate (OAA). Two C atoms are received as an acetyl unit, and oxidation produces two molecules of CO2. There are four oxidation- reduction steps which yield H+ and electrons to reduce NAD+ and FAD to NADH.H+ and FADH2
respectively. There are 8 intermediates in the cycle and one 4-C unit i.e. OAA, is regenerated.
Fig 5.3.2.The reactions of the citric acid (TCA) cycle. Refer to the text for details. (Source: Nelson and Cox, 2005, p 607 fig 16-7)
Reaction 1: Conversion of acetyl-CoA to citrate (∆G’o = -31.5 kJ/mol)
Acetyl-CoA (2-C) and the keto-acid oxaloacetate (4-C), undergo condensation to form citrate (6- C) in a reaction catalyzed by citrate synthase. On binding OAA, the enzyme undergoes a remarkable conformational change which facilitates attachment of its 2nd substrate, acetyl-CoA.
The reaction involves an aldol condensation followed by a hydrolysis in which H2O is used. The equilibrium for the hydrolysis lies far to the right so that the overall reaction moves towards the synthesis of citrate, driving the cycle forward.
Reaction 1 is irreversible under cellular conditions.
Reaction 2: Isomerization of citrate to isocitrate (∆G’o = ~5 kJ/mol)
Citrate is channeled directly to the active site of the next enzyme, aconitase, to undergo a two-step reaction that converts it to isocitrate (ICA). Aconitase carries 3 different Fe:S clusters which bind citrate and enable the reaction.
Fig 5.3.3. An Iron-sulfur center (pink) in aconitase which binds citrate (blue). ‘B’ is a basic residue in the enzyme which helps to position the substrate for binding and catalysis.
(Source: Nelson and Cox, 2005, p 610 fig 16-10)
The symmetrical citrate molecule is acted on asymmetrically by aconitase so that an –OH is shifted from C-3 in citrate and attached to C-4, a carbon atom derived originally from OAA. The reaction involves a dehydration followed by a hydration, with cis-aconitate as intermediate.
Though the aconitase reaction is reversible, it is pulled to the right by the rapid utilization of its product, ICA, in the next step.
Reaction 3: Oxidative decarboxylation of isocitrate to α-ketoglutarate (∆G’o = -21 kJ/mol) Isocitrate (6-C) is irreversibly oxidized and decarboxylated to α-ketoglutarate (5-C) in a two-step reaction catalyzed by isoctrate dehydrogenase (IDH). The enzyme uses NAD+ as a reductant and requires Mn2+. NADH.H + is produced in the the 1st step. Enzyme-bound intermediate, oxalosuccinate, is an unstable β-keto acid which loses CO2.
The rate of formation of α-ketoglutarate (α-KGA) is important in determining the overall rate of the TCA cycle.
Reaction 4: Oxidative decarboxylation of α-ketoglutarate to succinyl-CoA (∆G’o = -33 kJ/mol)
This reaction is catalyzed by a multi-enzyme complex, α-ketoglutarate dehydrogenase complex (α-KGA-DH complex), which functions in a manner identical to the PDH complex for pyruvate.
The α-KGA-DH complex and the PDH complex are homologous enzyme assemblies with the main function of transferring a keto group to CoA. The 1st and 3rd enzymes here, α-ketoglutarate dehydrogenase (α-KGA-DH) and trans-succinylase respectively, are functionally similar to E1 and E3 of the PDH complex. The 2nd enzyme, dihydrolipoyl dehydrogenase, is identical in both complexes. Oxidative decarboxylation of α-KGA yields succinyl-CoA (C-4), CO2 and NADH.H + . The overall reaction is exergonic and irreversible. The energy released is conserved in the thioester bond of succinyl-CoA (∆G’o for hydrolysis of succinyl-CoA = -36 kJ/mol).
Reaction 5: Conversion of succinyl-CoA to succinate (∆G’o = -2.1 kJ/mol)
Succinyl-CoA synthetase (= succinate thiokinase) converts succinyl-CoA to succinate, a 4-C compound. The reaction is a substrate-level phosphorylation in which the energy released on breaking the thioester bond of succinyl-CoA, is coupled to the phosphorylation of GDP/ADP to GTP/ATP respectively (∆G’o = -2.9 kJ/mol). Animal cells generally use GDP though some (e.g.
liver) have isozymes for both GDP and ADP. (Plant cells and bacteria use only ADP). The terminal phosphate group of GTP is transferred to ADP by nucleoside diphosphate kinase.
kinase nucleoside diphosphate
+
GT P ADP G DP + AT P
(∆G’o = 0 kJ/mol) Fig 5.3.4. Phosphorylation of ADP with GTP
Reaction 5 is reversible. The intermediates formed in subsequent reactions of the cycle, viz.
fumarate, malate and OAA, are 4-C compounds.
Reaction 6: Conversion of succinate to fumarate (∆G’o = +6 kJ/mol)
Succinate dehydrogenase (SDH) oxidizes succinate to fumarate, while its co-factor, FAD, is reduced to FADH2. SDH is embedded in the inner mitochondrial membrane (all other dehydrogenases are in the matrix) and transfers hydrogen and electrons directly from the substrate to FAD, without involving NAD+. The enzyme also has 3 different kinds of Fe:S clusters through which electrons flow from FADH2 to the ETS.
Reaction 7: Hydration of fumarate to malate (∆G’o = -3.4 kJ/mol)
Fumarase adds –H and –OH in the trans position across the double bond of fumarate, converting it to L-malate.
Reaction 8: Regeneration of oxalacetate (∆G’o = +29.7 kJ/mol)
Malate is oxidized to OAA by malate dehydrogenase (MDH). Its coenzyme NAD+ is reduced to generate the 3rd molecule of NADH.H + in the cycle. Regenerated OAA is now ready to accept another molecule of acetyl-CoA for the next round of the cycle.
The equilibrium favors malate formation over OAA, but is driven forward by the strongly exergonic condensation of OAA with acetyl-CoA in the next turn of the cycle, and the continuous re-oxidation of NADH.H+ in the ETS.
Overall reaction of one turn of the TCA cycle
Acetyl-CoA + NAD+ + FAD + GDP + Pi + H2O à 2 CO2 + 3 NADH + FADH2 +GTP + 2H+ + CoA
The citric acid cycle completely catabolizes acetyl-CoA. A tally of C, H and O atoms which enter the cycle with those that leave the cycle is as follows:
Carbon atoms:
Entry as 1 CH3.CO.SCoA = 2 Release as 2 CO2 = 2
Hence: Carbon atoms entering the cycle are fully oxidized Hydrogen atoms
:
Entry as 1 CH3.CO.SCoA = 3 Entry as 2 H2O = 4
Entry from 1 Pi (H3PO4) = 2
Total = 9 Hydrogen atoms enter the cycle Released as 3 NADH.H+ = 6
Released as 1 FADH2 = 2 Released as 1 CoASH = 1
Total = 9 Hydrogen atoms are released from the cycle Oxygen atoms
:
Entry as 1 CH3.CO.SCoA = 1 Entry as 2 H2O = 2
Entry from 1 Pi (H3PO4) = 1
Total = 4 Oxygen atoms enter the cycle Release as 2 CO2 = 4
Total = 4 Oxygen atoms are released from the cycle
In each turn of the cycle the C atoms derived from acetyl-CoA have definite locations in the molecules from citrate to succinyl-CoA, but the positions are variable in the later intermediates of the cycle. During the conversion of succinyl-CoA to succinate, there is randomization in the order of carbon atoms. Consequently, the two atoms that leave as CO2 in a particular turn of the cycle, are not the same two carbon atoms that had entered as acetyl-CoA in that turn. Additional turns of the cycle are required to remove these carbon atoms as CO2.
Water is the ultimate source for the H atoms not derived from acetyl-CoA, and of the oxygen which is used to produce CO2, though its components, H and O, are utilized indirectly.
Since there is no net removal of OAA in the TCA cycle; hence one molecule of OAA can theoretically bring about the oxidation of an infinite number of acetyl groups. In practice, however, the supply of OAA needs to be replenished, since it is used by other metabolic pathways as well.
REVERSIBILITY OF THE CYCLE
All the reactions of the cycle are reversible except those catalyzed by citrate synthase, IDH and α- ketoglutarate dehydrogenase. These reactions have a sufficiently large ∆G value to be irreversible and account for an overall ∆Go’ of -40 kJ/mol. Hence the cycle operates unidirectionally.
ENERGY YIELD
The step-wise degradation and oxidation of acetyl-CoA in the citric acid cycle is an efficient process for extraction of energy. The available energy is not released in one burst; it is transferred step-by-step to small packets of NADH and FADH2 to be carried to the electron transport system (ETS).
In every turn of the cycle involving one molecule of acetyl-CoA, 3 hydride ions (i.e. 6 electrons) are transferred to 3 NAD+ molecules while one pair of hydrogen atoms (i.e. 2 electrons) are transferred to an FAD molecule. Re-oxidation of 3 NADH and 1 FADH2 by oxidative phosphorylation in the ETS generates (7.5 +1.5) ATP i.e. 9 ATP (recall that ATP yield is 2.5/
NADH and 1.5/FADH2 if source is mitochondrial). In addition 1 GTP/ATP has also been obtained.
Hence, for every turn of the citric acid cycle:
TOTAL YIELD IS 10 ATP PER MOLECULE OF ACETYL COA
THE FINAL BALANCE SHEET
From Sections 1, 2 and 3 of this chapter we can now summarise the energy yield in terms of ATP when one molecule of glucose undergoes complete oxidation to CO2 and water:
Gain of ATP by substrate-level phosphorylation:
• Glycolysis = 4 ATP
• Citric acid cycle = 2 ATP Gain by oxidative phosphorylation:
• 2 NADH (cytosolic) in glycolysis = 3/5 ATP (depends on use of glycerophosphate/malate shuttle)
• 2 NADH (mitochondrial) by PDH complex = 5 ATP
• 6 NADH (mitochondrial) from TCA cycle = 15 ATP
• 2 FADH2 from TCA cycle = 3 ATP
Thus, total yield = 32/34 ATP per molecule of glucose oxidised Loss by utilization in glycolysis = 2 ATP
Hence:
NET ENERGY YIELD IN AEROBIC OXIDATION = 30/32 ATP PER MOLECULE OF GLUCOSE
Compare this to the net energy yield of a mere 2 ATP per molecule of glucose oxidized anaerobically.
It is estimated that by synthesizing ATP, the efficiency of trapping the energy released in glucose oxidation is 65%, This is calculated on the basis of actual free energy changes that occur in cellular conditions of pH, and low, unequal concentrations of ATP, ADP and Pi.
PROVISION OF SUBSTRATES FOR THE TCA CYCLE
Essential substrates for the cycle are acetyl-CoA (2-C) and oxaloacetate (4-C).
Acetyl-CoA is required in stoichometric amounts for each turn of the cycle and is obtained from:
• Glucose – as explained in sections 1 and 2
• Fatty acids – by β-oxidation (refer Ch 6)
Normally, OAA is present in the cell in very low concentrations. Since it is regenerated in every round of the cycle, OAA is required only in catalytic amounts. However, the TCA cycle may become deficient in its OAA supply if this keto-acid is pilfered by other metabolic pathways like gluconeogenesis and transamination. In order to keep the cycle operating at a steady rate, the system takes the help of anaplerotic reactions which replenish OAA, or other cycle intermediates.
pyruvate carboxylas e
+
Pyruvate H CO3_ + AT P O A A + ADP + P i ( liver)
O A A+GT P( muscle)
+ CO2 + G DP PEP
PEP carboxy kinase
carboxylase PEP
yeast
,
bacteria
,
higher plants
( )
+
O A A P i PEP+ H CO3
_
malic enzyme
N A D +
( P)H.H
+ +
Pyruvate H CO3_ Malat e+ N A D( P)+
+ N A D( P)++
Glutamic
acid H O2 dehydrogenase a -K GA+ N A D( P)H.H++NH3 glutamate
Fig 5.3.5. Anaplerotic reactions that replenish intermediates of the TCA cycle. ( PEP = Phosphoenolpyruvate;
OAA = Oxaloacetate; a-KGA = α-Ketoglutarate)
The pyruvate carboxylase reaction is one of the most important anaplerotic reactions. Pyruvate carboxylase is allosterically stimulated by acetyl-CoA and concomitantly increases production of OAA from pyruvate when [acetyl-CoA] becomes high. Succinyl-CoA is produced by oxidation of odd-chain fatty acids (see Chapter VI), and the catabolism of amino acids like Ileu, Met and Val.
Transamination and deamination reactions of amino acids are reversible and depending on metabolic demand, they also replenish cycle intermediates (see Chapter VII).
The Glyoxylate cycle is an alternative pathway, which processes isocitrate without decarboxylations, and can provide succinate, OAA and other intermediates of the TCA cycle. It occurs in germinating seeds and some microbes. Unlike the TCA cycle, the glyoxylate cycle enables net conversion of acetyl-CoA into 4-C intermediates.
Four C atoms enter the cycle as two molecules of acetyl-CoA and four C atoms leave as succinate.
Only one molecule of NADH.H+ is produced per turn of the cycle.
H2O
e citrate Iso
Succinat e lyase isocitrat
_
C OO -
C
H O
GLYOXYLATE Malat e
N A D+ H.H+ N A D
Acetyl -CoA
CoA
OA A Citrat e
malate synthas e
Acetyl -CoA
CoA
H2O
Fig 5.3.6. The glyoxylate cycle in plant glyoxysomes.
ROLE OF VITAMINS
Four vitamins of the B-complex group are essential constituents of co-factors used in the TCA cycle:
• Riboflavin – for FAD, used by α-KGA-DH, SDH and also PDH
• Niacin – for NAD, used by IDH, α-KGA-DH, MDH and also PDH
• Thiamin (TDP) – used by α-KGA-DH and also PDH
• Pantothenic acid – is a part of Coenzyme A which carries the acetyl and succinyl groups
ACTION OF INHIBITORS
Malonate competes with succinate and inhibits SDH. Accumulation of succinate in a malonic- inhibited system stops the TCA cycle. Tissue preparations inhibited by malonate, continue with the cycle if stoichometric amounts of fumarate/malate or OAA are added but not if the tricarboxylic acids or α-KGA are provided.
Fluoroacetate combines with OAA to form fluorocitrtate, which inhibits aconitase. Citrate accumulates in such a system and the TCA cycle is suspended.
Arsenite complexes with –SH group of lipoamide to inhibit α-KGA-DH complex so that α-KGA accumulates and the cycle is stopped.
METABOLIC ROLE OF THE TCA CYCLE
The TCA cycle has a pivotal role in metabolism. It is essentially amphibolic and is connected to several catabolic as well as anabolic pathways. The 8 intermediates in the cycle are links to metabolites in other metabolic pathways.
Catabolic role:
The citric acid cycle oxidizes carbohydrates, lipids and amino acids. The 2-C, 4-C and 5-C end- products of other catabolic processes undergo degradation when fed into the cycle as acetyl-CoA,
succinyl-CoA or any of the other intermediates. The C-skeleton is oxidized to CO2 while the hydrogen is finally passed on to molecular oxygen to form water. We take below an example of the amino acid Isoleucine:
-CoA
Propionyl
+
Acet yl CoA-+
CO2 Isoleucine( 6-C)2CO2
TC A cycl e
Pyruvate
-CoA Succinyl CO2
TC A cycl e OA A
PEP
+
CO2 PEP carboxy kinasepyruvate kinase
2CO2 TC A cycl e
PDH compl xe
Acet yl CoA-
+
CO2
Fig 5.3.7. Catabolic role of the TCA cycle as shown in the metabolism of the carbon skeleton of Isoleucine. The C skeleton is traced in red (OAA = oxaloacetate; PDH complex = pyruvate dehydrogenase complex; PEP = phosphoenol pyruvate)
Anabolic role
Purines
, Arg
Gln Pro,
Glu Fatty acids sterols,
Tyr Phe Cys, , Ser Gly Try, ,
Glucose
PEP
Porphyrins Heme Acetyl -CoA
CYCLE CITRIC ACID
SUCCINYL-
CoA
CITRATE OA A
a-KGA Pyrimidines
Asp,Arg
Fig 5.3.8. Schematic diagram of the anabolic role of the TCA cycle. Abbreviated names of the amino acids have been used: PEP = Phosphoenolpyruvate; OAA = Oxaloacetate; a-KGA = α-Ketoglutarate. (Adapted from Nelson and Cox, 2005, p 617 fig 16-15)
The cycle is a major source of precursors for many biosynthetic pathways:
• Gluconeogenesis – all major acids from citrate to OAA can undergo gluconeogenesis to form glucose. The key enzyme required is PEP-carboxykinase. The acids enter the TCA cycle from various sources, the chief one being OAA from pyruvate (using pyruvate carboxylase). The products leave the mitochondrion via malate for gluconeogenesis in the cytosol. (refer Section 6)
• Synthesis of amino acids – the C-skeleton of cycle intermediates are a source for synthesis of non-essential amino acids. Transamination and deamination reactions are reversible and ideal for this purpose.
Reversal of the glutamate dehydrogenase reaction:
+ N A D( P)++
Glutamic
acid H O2
H N 3
+ +N A D( P)H.H+
a -K GA
dehydrogenase glutamate
Transaminations:
glutamate amino transferase Alanine
+
a -K GA Glutamat e+Pyruvate
OA amino transferase Aspartat e +Pyruvate
alanine Alanine
+
A
Fig 5.3.9. Synthesis of amino acids from intermediates of the TCA cycle.
Fatty acid and cholesterol synthesis – is linked to citrate of the TCA cycle. The enzymes for fatty acid synthesis are located in the cytosol. A citrate transporter conveys mitochondrial citrate to the cytosol where the enzyme ATP-citrate lyase cleaves it to provide acetyl-CoA for fatty acid synthesis
Fig 5.3.10. TCA cycle and fatty acid synthesis.
(Source: Murray et al, 2003, p 135 fig 16-5)
• Purine and pyrimidine nucleotides – are synthesized from α-KGA and OAA
• Porphyrin ring of heme groups – are synthesized from succinyl-CoA and used in synthesis of hemoglobin, myoglobin and cytochromes
REGULATION OF TCA CYCLE
The TCA cycle is at the junction of many major metabolic pathways and regulation of its activity is crucial to the well-being of an aerobic organism. The rate of the TCA cycle is primarily geared to the energy needs of the cell at any particular point in time. The regulation of the PDH complex, as also that of the –oxidation of fatty acids, are major determinants of the rate of provision of acetyl-CoA to the cycle. The factors that regulate the TCA cycle and the PDH complex are similar in many respects so that the two processes are co-ordinated.
a- H
complexKGA D-
NA DH
-CoA succinyl
_
+ C a2+ I DH
-CoA succinyl
NA DH A T P,
_
,C a2+ ADP + CITRATE
FUMARATE
MALATE ISOCITRATE
a-KGA
SUCCINATE
SUCCINYL-
CoA A A
O fatty acids N A DH,
A T P,acet yl CoA- ,
_
C a2+ ,
CoA N A D+, ,
A M P +
Pyruvate P HD complex Acet yl CoA-
citratesyn thase
P A D + NA DH,succinyl-CoA
citrate A T P,
_
DH O A A S
_
Fig 5.3.11. Regulators of the citric acid cycle: green signs indicate stimulation and red signs indicate inhibition.
(PDH = pyruvate dehydrogenase; IDH = isocitrate dehydrogenase ; α-KGA-DH = α-ketoglutarate dehydrogenase; SDH = succinate dehydrogenase)
The rate-determining steps are the 3 exergonic reactions catalyzed by citrate synthase (CS), IDH and α-KGA-DH. The mechanism of regulation depends on local molecules in the cell, many of which are part of the cycle itself. Regulation is therefore quick, effective and in tune with the existing needs of the cell. Surprisingly, covalent modification of the regulatory enzymes by the phosphorylation-dephosphorylation cycle, does not take place. In addition, hormones have no direct role in regulating the cycle.
There are three fairly simple means by which the TCA cycle is regulated:
o Substrate availability o End-product inhibition
o Competitive feedback inhibition
Substrate availability is a key factor in determining whether the cycle can fulfill its purpose. The supply of acetyl-CoA, OAA and NAD+, is particularly important. The supply of acetyl-CoA is determined by the activity of the PDH complex and β-oxidation of fatty acids, while the continued availability of OAA is ensured, if necessary, through anaplerotic reactions. Thus, if [acetyl-CoA]
is high but availability of OAA is limited (by diversion into other metabolic pathways), then high [acetyl-CoA] stimulates pyruvate carboxylase allosterically to increase OAA production from pyruvate. When the rate of glycolysis is greater than the rate of the TCA cycle, accumulating citrate inhibits PFK-1. In this way, the production and utilization of acetyl-CoA is balanced so that the TCA cycle can proceed at the required rate. If TCA is inhibited at some other step (e.g. by NADH), increased OAA does not accelerate the cycle; instead OAA equilibrates with malate and is transported out of the mitochondrion.
End-product inhibition is a direct inhibition of an enzyme by accumulation of the products of the reaction catalyzed by it. All three regulatory enzymes of the TCA cycle are inhibited in this way by mass action. Thus:
• CS is inhibited by high [citrate]
• IDH is severely inhibited by increased [NADH]
• α-KGA-DH is severely inhibited by high [NADH] and by [succinyl-CoA]
The concentrations of the end-products not only affect their own specific enzymes but also result in a “domino” effect on other enzymes.
Feedback inhibitions are caused by intermediates which are products of later reactions. Thus, CS and IDH are inhibited by succinyl-CoA, which is a product of a subsequent step of the cycle.
Succinyl-CoA competes with OAA for citrate synthase. Inhibition of CS by NADH, and of IDH by ATP are also examples of feedback inhibition.
Some of the molecules regulating the enzymes are allosteric modulators:
• high [ADP] – activates CS and IDH
• high [ATP] – inhibits CS and IDH
• Ca2+ release – which signals muscle contraction, stimulates PDH, IDH and α-KGA-DH so that increase in rate of TCA cycle makes more ATP available for muscle contraction
• Long-chain acyl CoA – inhibit CS
The rates of all the reactions producing NADH are dependent on the availability of NAD+, which in turn depends on re-oxidation of NADH in the respiratory chain. High ratio of [NADH]/[NAD+] inhibits all three regulatory enzymes of the cycle as well as the PDH complex.
The rate of the TCA cycle is closely linked to glycolysis and the electron transport chain.
Glycolysis also responds to the cellular levels of ATP, NADH and citrate, so that the provision of pyruvate matches the rate of its utilization in the TCA cycle. When the energy state of the cell is high, the [ADP] available is low. This reduces the rate of oxidative phosphorylation and hence, the rate of re-oxidation of NADH. The consequent reduction in the supply of NAD+ decreases the rate of the TCA cycle. Thus, the TCA cycle is under “respiratory control”.
Evidence now shows that some enzymes of the citric acid cycle may be associated as supramolecular complexes (metabolons) which enable more efficient substrate channeling during reaction sequences. Obviously mechanisms regulating the citric acid cycle need further elucidation.