Download AKA TCA CYCLE, KREB`S CYCLE

Note Set 12
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CITRIC ACID CYCLE (AKA TCA CYCLE, KREB'S CYCLE)
2nd pathway involved in the complete aerobic oxidation of glucose to CO2 and H 2O
Focus:
1. Conversion of pyruvate to acetylCoA
an obligatory step in the aerobic oxidation of glucose
2. The reactions of the TCA cycle
a catalytic process by which acetylCoA is degraded to CO2 and H 2O with
accompanying reduction of NAD+ to NADH
3. Regulation of the TCA cycle
4. Anabolic function of the TCA cycle
GENERAL CONSIDERATIONS
1. lactate formation from glucose has a ∆ G°' = -47 kcal/mol; the complete oxidation of
glucose to CO2 and H 2O has a ∆ G = -686 kcal/mol
•aerobic organisms trap this extra energy in the form of ATP
the yield of ATP is about 19 times greater than in glycolysis
2. the aerobic oxidation of glucose involves 3 separate enzymatic pathways:
glycolysis
TCA cycle
terminal respiratory chain
3. the TCA cycle functions mainly to oxidize acetylCoA formed from pyruvate to CO2
and H 2O
this oxidation process is coupled to the reduction of NAD+ to NADH
4. all the enzymes of the TCA cycle except one are located in the matrix compartment
of the mitochondria
all except 1 are water soluble proteins
HISTORICAL BACKGROUND
1. main outline of the cycle was worked out in a relatively short period of time (19351937)
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2. a key finding by A. Szent-Gyorgi was that addition of malate or oxaloacetate to
muscle homogenates catalyzes the uptake of oxygen in amounts greater than
expected from the amount of acid added (acid = malic acid, oxaloacetic acid...etc)
•he postulated the following sequence:
succinate--->fumerate--->malate--->oxaloacetate
•during the same period Martius and Knoop showed the following reactions:
citrate--->α-ketoglutarate--->succinate
3. the complete cycle was detailed by Krebs:
•the key observations were:
stimulation of glucose oxidation by di- and tricarboxylic acids, eg: pyruvate,
succinate, citrate
inhibition of oxidation of pyruvate by malonate, an inhibitor of succinate
dehydrogenase
formation of citrate from "a substance derived from pyruvate" and oxaloacetate
in a malonate poisoned system, the oxidation of pyruvate could be relieved by
addition of large amounts of oxaloacetate
4. even though almost all the reactions of the TCA cycle were known by 1937, the
mechanism by which pyruvate and oxaloacetate react to form citrate was not
elucidated until 1950, mainly through the work of Reed
SYNTHESIS OF Acetyl CoA FROM DECARBOXYLATION OF PYRUVATE
oxaloacetate reacts with acetylCoA
formed by an oxidative decarboxylation of pyruvate
very important to TCA cycle, although not part of the cycle itself
1. Synthesis of adetyl CoA from pyruvate is catalyzed by the pyruvate dehydrogenase
complex, a multiprotein enzyme complex
MW 7 x 107
2. overall reaction:
pyruvate + CoA + NAD+ ----->acetylCoA + CO2 + NADH + H+
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∆ G°' = -8 kcal/mol
3. components of the complex:
•24 units of pyruvate dehydrogenase (TPP coenzyme, TPP = thiamine
pyrophosphate))
•24 units of dihydrolipoyl transacetylase
lipoic acid covalently bound
linked through a peptide bond with the ε -amino group of a lys
•12 units of dihydrolipoyl dehydrogenase (NAD+ coenzyme)
•complex also contains 6 units of pyruvate dehydrogenase kinase and 2 units of
phosphatase
catalyze the phosphorylation and dephosphorylation of a serine on pyruvate
dehydrogenase (PDH)
important for regulation
4. regulation of the complex
•if [ATP] is high, and Mg++ is low, and NADH is high, the kinase phosphorylates the
ser on PDH and the enzyme is inactivated
•if [ATP] is low, and Mg++ and Ca ++ are present, the phosphatase removes the Pi
from ser and this activates the enzyme
•Therefore:
When the intracellular pool of ATP/NADH is high, activity of the complex is
suppressed
When the ATP pool is low, and Mg ++ (and/or Ca ++ ) are high, the complex is
activated and pyruvate utilization is increased
Note that hormones can activate PDH by increasing Ca++ release into the cytosol
and/or mitos
REACTIONS OF THE TCA CYCLE
•cyclic process involving participation of 8 enzymes
•all localized in the mitochondria
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with the exception of succinate dehydrogenase, which is membrane bound,
remaining 7 enzymes are soluble proteins present in the matrix compartment
of the mitos
•it is an autocatalytic process in which 2 C's of acetate are put in and 2 C's are
oxidized to 2 CO2's in one turn of the cycle
•there are 4 oxidation steps
•3 result in the reduction of NAD+ to NADH
•1 of FAD to FADH2
•there is one substrate level phosphorylation step resulting in the esterification of
GDP and Pi
OVERALL REACTION:
CH3-C-SCoA + 3NAD+ + FAD + GDP + Pi + 2H2O -----> 2CO2 + CoA(SH) + 3NADH +
3H+ + FADH2 + GTP
DETAILED REACTIONS:
1. formation of citrate
•citrate (6C) is formed from oxaloacetate (4C) and acetyl CoA (2C)
•catalyzed by citrate synthase (condensing enzyme)
•exergonic reaction
∆ G°' = - 7.7 kcal/mol
•aldol condensation followed by a hydrolysis
•proceeds at expense of energy of hydrolysis of the thioester bond of acetyl CoA
•citryl CoA is formed as an intermediate
•rate of reaction depends on [acetyl CoA] and [oxaloacetate]
•inhibited by succinyl CoA and ATP
•important control step in the cycle
2 and 3. isomerization of citrate to form isocitrate
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•catalyzed by aconitase
•iron-sulfer protein (nonheme iron protein)
•equilibrium mixture consists of 93% citrate and 7% isocitrate
•1st step is a dehydration
∆ G°' = + 2 kcal/mol
•2nd step is a hydration
∆ G°' = - 0.5 kcal/mol
•overall, an H and an OH switch positions
4. formation of α -ketoglutarate from isocitrate
•catalyzed by isocitrate dehydrogenase (NAD+ dependent)
•oxidative decarboxylation
∆ G°' = - 2 kcal/mol
•isocitrate is oxidized to oxalosuccinate
•NAD+ is reduced to NADH
•oxalosuccinate is decarboxylated to α-Ketogluterate (α -KG)
decarboxylation step requires Mg++
in the absence of divalent metal oxalosuccinate accumulates
•isocitrate dehydrogenase shows allosteric control with ADP
ADP activates
ATP and NADH inhibit
KM for isocitrate decreases as a function of increasing [ADP]
5. formation of succinyl CoA from α -ketoglutarate
•the 2nd oxidative decarboxylation
•catalyzed by the
-ketoglutarate dehydrogenase complex
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similar to the pyruvate dehydrogenase complex
mechanism is similar, but no kinase or phosphatase regulation
∆ G°' = - 7.2 kcal/mol
MW = 2.7 x 106
•consists of 3 enzymes
α -KG dehydrogenase (TPP)
dihydrolipoyl transsuccinylase (lipoic acid)
dihydrolipoyl dehydrogenase (FAD)
6. formation of succinate
•catalyzed by succinyl CoA synthetase
∆ G°' = - 0.8 kcal/mol
•substrate level phosphorylation
GDP is phosphorylated to GTP
only step that directly yields a high energy P bond
•energy of hydrolysis of the thioester bond (∆ G°' = -7.5 kcal/mol) is conserved in the
form of the terminal P of GTP
•mechanism involves a phosphorylated enzyme intermediate (hisP)
7. succinate is oxidized to fumarate
•succinate dehydrogenase
∆ G°' ≈ 0
•contains covalently bound FAD
FAD gets reduced to FADH2
∆ G°' not enough to reduce NAD+ because 2 Hs that are attached to Cs are
removed…this requires a lot of energy
• contains iron-sulfer protein (will be discussed in connection with electron transport)
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•membrane bound
8. fumarate is converted to L-malate
•by fumarase
∆ G = - 0.9 kcal/mol
•trans addition of water to fumarate yielding L-malate
9. oxaloacetate is regenerated by oxidation of L-malate
•malate dehydrogenase
∆ G°' = + 7.1 kcal/mol
•NAD+ is reduced to NADH
•inhibited by oxaloacetate
•even though reaction is endergonic, products are rapidly removed and reaction
proceeds in forward direction
REGULATION OF THE TCA CYCLE
1. Modulators of enzyme activity
•includes ADP, ATP NADH, citrate, succinyl CoA, metals, etc.
•most important is probably the relative concentration of ATP and ADP
at high ATP/ADP forward flux is inhibited
at low ATP/ADP forward flux is stimulated
•enzymes regulated by ATP and ADP:
Pyruvate Dehydrogenase Complex: phosphorylation by kinase to inactive form
occurs at high ATP/ADP
Citrate synthase is inhibited by ATP
Isocitrate dehydrogenase is stimulated by ADP
2. Respiratory Control
•forward flux of cycle depends on availability of NAD +
•NAD+ depends on rate of reoxidation of NADH
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•oxidation of NADH is under respiratory control
rate is high when ATP/ADP is low
•oxidation of succinate is also under respiratory control
ENERGETICS
•ATP yield in aerobic utilization of glucose:
NADH
ATP
FADH2
glycolysis
Glucose à 2 pyruvate
2
2
0
PDH complex
2 pyruvateà 2 acetyl CoA
2
0
0
TCA cycle
2 acetyl CoA à 4 CO2
6
2 GTP
2
•in the terminal respiratory chain:
the oxidation of each NADH is coupled to the synthesis of about 3 ATP
the oxidation of FADH2 is coupled to the synthesis of about 2 ATP
GTP can transfer its γ phosphate to ADP to form ATP catalyzed by nucleoside
diphosphate kinase
total ATP yield is thus about 38 ATP's formed/mol of glucose oxidized
•glucose à CO2 + H2O
∆ G°' = - 686 kcal/mol
net yield of ATP = about 38
•conservation of energy = 38 x 7.3 = 277 kcal/mol
•efficiency = 277/686 = 40%
ANAPLEROTIC REACTIONS
1. In addition to its catabolic function, TCA cycle also provides important intermediates
in anabolism (biosynthesis)
•a number of amino acids are made from TCA cycle intermediates
2. Reactions that replenish TCA cycle intermediates:
•pyruvate carboxylase reaction
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oxaloacetate can be made from pyruvate
[oxaloacetate] is usually low because Keq of citrate synthase favors formation of
citrate
if [oxaloacetate] gets too low, it is replenished by carboxylation of pyruvate catalyzed
by pyruvate carboxylase
pyruvate carboxylase is a biotin enzyme
pyruvate carboxylase is an allosteric enzyme that is stimulated by high [acetyl CoA]
•malic enzyme
pyruvate + CO2 + NADPH + H+ ----> L-malate + NADP +
GLYOXYLATE CYCLE
An anabolic variant of citric acid cycle
1. plant cells (and some microorganisms) can carry out net synthesis of carbohydrate
from fat
•crucial to develpoment of seeds
energy stored as triacylglycerols
•vegetable oils we use: mixtures of triacylglycerols from seeds
•when seeds germinate, triacylglycerol broken down, converted to sugars
energy and raw material for growth
2. glyoxylate cycle allows net synthesis of carbohydrate from acetyl CoA
•2 main fates of acetyl CoA are oxidation thru TCA, and synthesis of fatty acids
•acetyl CoA cannot be converted to pyruvate
PDH complx reaction is irreversible
acetyl CoA can be converted to oxalo., but 2 C's lost as CO 2 in process, so it is not
net synthesis
3. glyoxylate cycle converts 2 acetyl units, as acetyl CoA, to succinate
•uses some TCA cycle enzymes
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•bypasses 2 reactions where C is lost
IDH and α KGDH
•2nd mole of acetyl CoA brought in at bypass
•each turn of cycle brings in 2 2C units and results in net synthesis of 1 4C unit
(succinate)
•succinate transported to mito where it is converted to oxaloactate via TCA
•acetate can be used as C source via glyoxylate cycle:
1st converted to acetyl CoA by acetate thiokinase
acetate + CoA-SH + ATP ----> acetyl CoA + AMP + PPi
4. glyoxylate cycle and also some of the β oxidation of FAs (fatty acids) takes place in
glyoxysomes
β oxidation generates the acetyl CoA for glyoxylate cycle
5. reactions:
•acetyl CoA à isocitrate as in TCA
enzymes are isozymes of TCA enzymes, specialized for glyoxylate cycle
then:
•isocitrate cleaved to succinate and glyoxylate by isocitrate lyase
succ goes to TCA in mitos
•glyoxylate accepts acetate from another acetyl CoA to form malate: catalyzed by
malate synthase
•malate then to oxaloactate for another turn by isozyme of malate dehydrogenase
PENTOSE PHOSPHATE PATHWAY
1. an alternative pathway for carbohydrate oxidation
•also called hexose monophosphate shunt
•operates to varying extents in different cells and tissues
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-primarily anabolic
-under some conditions can oxidize glucose completely to CO2
2. 2 primary functions:
•to provide NADPH for reductive biosynthesis
•to provide ribose-5-P for nucleotide and nucleic acid biosyn.
•additional function:
-metabolizes dietary pentose sugars from digestion of nucleic acids
3. NADPH
•same as NADH, but w/ 2' P on one of the ribose groups
•metabolically different:
-NAD+/NADH pair function w/enzymes that oxidize substrates
-NADP+/NADPH pair function with enzxymes that reduce substrates
•use: for example, for fatty acid and steroid biosynthesis
-adrenal gland, liver, adipose tissue, mammary gland have high PP pathway activity
-also is ultimate e- source for reduction of ribonucleotides to deoxyribonuccleotides, so
rapidly growing tissue has high PP pathway enzymes
4. PP pathway located in cytoplasm
A. Oxidative Phase
•generation of reducing power as NADPH
1. conversion of glucose-6-P to ribulose-5-P
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-1st and 3rd are oxidative reactions
•1st: glucose-6-P dehydrogenase
glu 6-P to 6-phosphogluconolactone
•converted to 6-phosphogluconate by lactonase
•converted to ribulose-5-P by 6-phosphogluconate dehydrogenase
CO2 lost
2. net result:
•2 mol of NADPH
•1 mol pentose phosphate
•lose 1 CO2
B. Nonoxidative Phase
•fates of pentose phosphates tailored to metabolic needs
1. next ribulose-5-P converted to ribose-5-P by phosphopentose isomerase
•enediol intermediate
-like triose-P isomerase and phosphoglucoisomerase
•primary functions of pathway now fullfilled
NADPH and ribose-5-P
2. balanced equation for pathway so far:
glucose-6-P + 2NADP+ à ribose-5-P + CO2 + 2NADPH + 2H+
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3. many cells need the NADPH, but don't need all the ribose-5-P
•so there are ways to catabolize the extra ribose-5-P in a series of sugar phosphate
transformations
•net result:
3 5C sugar-P's converted to 2 6C sugar-P's and 1 3C sugar-P
-the 3C sugar-P is glyceraldehyde-3-P and it enters glycolysis
-the hexoses can be catabolized either by recycling through the PP pathway or entering
glycolysis
4. 3 enzymes involved in conversions:
phosphopentose epimerase (PPE)
transketolase (TK)
transaldolase (TA)
•PPE converts ribulose-5-P to xylulose-5-P (its epimer)
•x-5-P reacts w/ ribose-5-P to form glyceraldehyde-3-P and 7C sugar-P
sedoheptulose-7-P
-E is TK
-now must get rid of sed-7-P
•sed-7-P + glycer-3-P form fructose-6-P + erythrose-4-P
-E is TA
-net so far: 2 5C-sugarPs converted to 1 6C and 1 4C
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-now get rid of erythrose:
•x-5-P + erythrose-4-P form glyceraldehyde-3-P and fructose-6-P
-E is TK
•so far, input of 3 pentose P's required: 2 in 1st TK reaction, 1 in 2nd TK reaction
•so considering these 3 pass through oxidative phase, pathway to this point is:
-oxidative phase:
3 glucose-6-P + 6NADP+ à 3 pentose-5-P + 6NADPH + 6H+ + 3CO2
-plus rearrangement of nonoxidative phase:
3 pentose-5-P ••> 2 F-6-P + G-3-P
-overall:
3 glucose-6-P + 6NADP+ ••> 2 F-6-P + G-3-P3 + 6NADPH + 6H+ + 3CO2
•alternatively, can write equation for complete oxidation of 1 mol of hexose to CO2
6 hexose-6-P + 12NADP à 5 hexose-6-P + 6CO 2 + 12NADPH + 12H+
C. fate of sugarP's depends on metabolic needs of cell
1. nucleotide biosynthesis needed:
ribose-5-P and NADPH generated
2. NADPH needed for other reductive biosynthesis, don't need nucleotides:
G-3-P and F-6-P converted back to glu-6-P for reentry into PP pathway so more NADPH can be
made
3. Energy needed along with the NADPH:
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F-6-P and G-3-P go thru glycolysis and TCA
•completely oxidized to CO 2 and H 2O
•ATP generated
4. unlikely that any one of these modes operates exclusively in any one cell
•each cell has multiple metabolic needs
D. human genetic disorders involving PP pathway enzymes
1. deficiency of glucose-6-P dehydrogenase
•causes hemolytic anemia (massive destruction of red cells) when person under oxidative
stress (peroxides generated)
•peroxides normally inactivated via reduction by glutathione (reducing agent)
•also, Hb Fe kept in Fe+2 state by glutathione
•glutathione made frm cys, gly and γ glu
•oxidized glutathione is reduced by glutathione reductase
-requires NADPH
•PP pathway only source of NADPH in red cell
•mutant E abt 10% activity of wt
-OK until lots of peroxides show up
-then methemoglobin accumulates, changes structure of cell, membrane weakens, cells
rupture
*antimalarial
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2. Wernicke-Korsakoff syndrome
•mental disorder and loss of memory and partial paralysis
-from thiamine def. in affected people
•TK has 10 fold lower affinity for thiamine
-other TPP E's not affected
3. both diseases illustrate interplay between genetics and environment in onset of disease
•symptoms become apparent only after some kind of moderate stress that does not affect
normal individuals