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Chapt. 20 TCA cycle
Ch. 20 Tricarboxylic acid cyle
Student Learning Outcomes:
• Describe relevance of TCA cycle
• Acetyl CoA funnels products
• Describe reactions of TCA cycle in cell
respiration: 2C added, oxidations, rearrangements->
NADH, FAD(2H), GTP, CO2 produced
• Explain TCA cycle intermediates are used in
biosynthetic reactions
• Describe how TCA cycle is regulated by ATP
demand: ADP levels, NADH/NAD+ ratio
Overview TCA cycle
TCA cycle (Kreb’s cycle)
or citric acid cycle:
• Generates 2/3 of ATP
• 2C unit Acetyl CoA
• Adds to 4C oxaloacetate
• Forms 6C citrate
• Oxidations,
rearrangements ->
• Oxaloacetate again
• 2 CO2 released
• 3 NADH, 1 FAD(2H)
• 1 GTP
Fig. 1
II. Reactions of TCA cycle
Reactions of TCA cycle:
• 2 C of Acetyl CoA are oxidized to
CO2 (not the same 2 that enter)
• Electrons conserved through
NAD+, FAD -> go to electron
transport chain
• 1 GTP substrate level
phosphorylation:
• 2.5 ATP/NADH; 1.5 ATP/FAD(2H)
• Net 10 high-energy P/Acetyl group
Fig. 2
TCA cycle reactions
TCA cycle Reactions.
A. Formation, oxidation
of isocitrate:
2C onto oxaloacetate
(synthase C-C
synthetases need ~P)
Aconitrase move OH
(will become C=O)
Isocitrate Dehydrogenase
oxidizes –OH, cleaves
COOH -> CO2
also get NADH
Fig. 3**
TCA cycle reactions
TCA cycle Reactions.
B. a-ketoglutarate to
Succinyl CoA:
Oxidative decarboxylation
releases CO2
Succinyl joins to CoA
NADH formed
GTP made from
activated succinyl CoA
Fig. 3**
TCA cycle reactions
TCA cycle Reactions.
D. Oxidation of Succinate
to oxaloacetate:
2 e- from succinate
to FAD-> FAD(2H)
Fumarate formed
H2O added -> malate
2 e- to NAD+ -> NADH
Oxaloacetate restored
(common series of oxidations
to C=C, add H2O -> -OH,
oxidize -OH to C=O)
Fig. 3**
III. Coenzymes are critical: NAD+
• Many dehydrogenases use NAD+ coenzyme
• NAD+ accepts 2 e- (hydride ion H-): -OH -> C=O
• NAD+, and NADH are released from enzyme;
• Can bind and inhibit different dehydrogenases
• NAD+/NADH regulatory role (e-transport rate)
Fig. 5
III. Coenzymes are critical for TCA cycle
• FAD can accept e- singly (as C=C formation)
• FAD remains tightly bound to enzymes
Fig. 4
Fig. 6 membrane
bound succinate
dehydrogenase:
FAD transfers e- to
Fe-S group and to
ETC
Coenzyme CoA in TCA cycle
CoASH coenzyme forms thioester bond:
• High energy bond
(Fig. 8.12 structure of CoASH formed from pantothenate)
Fig. 7
Coenzymes CoASH; TPP
Coenzymes CoASH, TPP
(Figs. 8.11, 8.12)
Coenzymes in a-ketoacid dehydrogenase complex.
C. a-ketoacid dehydrogenase
complex:
• 3 member family (pyruvate dehydrogenase,
branched-chain aa dehydrogenase)
• Ketoacid is decarboxylated
• CO2 released
• Keto group activated, attached CoA
• Huge enzyme complexes
• (3 enzymes E1, E2, E3)
• Different coenzymes in each
Fig. 8
a-ketoacid dehydrogenase enzyme complex:
• 3 enzymes E1, E2, E3
• Coenzymes: TPP(thiamine pyrophosphate).
Lipoate, FAD
Fig. 9
Lipoate is a coenzyme
Lipoate coenzyme:
•
•
•
•
•
Made from carbohydrate, aa
Not from vitamin precursor
Attaches to –NH2 of lysine of enzyme
Transfers acyl fragment to CoASH
Transfers e- from SH to FAD
Fig. 10
Energetics of TCA cycle
Energetics of TCA cycle: overall net -DG0’
• Some reactions positive;
• Some loss of energy as heat (-13 kcal)
• Oxidation of NADH,
FAD(2H) helps pull
TCA cycle forward
Very efficient cycle:
• Yield 207 Kcal from
1 Acetyl -> CO2
• (90% theoretical 228)
• Table 20.1
Fig. 11
V. Regulation of TCA cycle
Many points of regulation of TCA cycle:
• PO4 state of ATP (ATP:ADP)
• Reduction state of NAD+ (ratio NADH:NAD+)
• NADH must enter ETC
Fig. 12
Table 20.2 general regulatory mechanisms
Table 20.2 general regulation metabolic paths
•
•
•
•
Regulation matches function (tissue-specific differences)
Often at rate-limiting step, slowest step
Often first committed step of pathway, or branchpoint
Regulatory enzymes often catalyze physiological irreversible
reactions (differ in catabolic, biosynthetic paths)
• Often feedback regulation by end product
• Compartmentalization also helps control access to enzymes
• Hormonal regulation integrates responses among tissues:
• Phosphorylation state of enyzmes
• Amount of enzyme
• Concentration of activator or inhibitor
Citrate synthase simple regulation
Citrate synthase simple regulation:
• Concentration of oxaloacetate, the substrate
• Citrate is product inhibitor, competitive with S
• Malate -> oxoaloacetate favors malate
•
•
If NADH/NAD+ ratio decreases, more oxaloacetate
If isocitrate dehydrogenase activated, less citrate
Allosteric regulation of isocitrate Dehydrogenase
Isocitrate dehydrogenase (ICDH):
• Rate-limiting step
• Allosteric activation by ADP
• Small inc ADP -> large change rate
• Allosteric inhibition by NADH
• Reflect function of ETC
Fig. 13
Other regulation of TCA
Regulation of a-ketoglutarate dehydrogenase:
• Product inhibited by NADH, succinyl CoA
• May be inhibited by GTP
• Like ICDH, responds to levels ADP, ETC activity
Regulation of TCA cycle intermediates:
• Ensures NADH made fast enough for ATP homeostasis
• Keeps concentration of intermediates appropriate
VI. Precursors of Acetyl CoA
VI. Many fuels feed directly into Acetyl CoA
• Will be completely oxidized to CO2
Fig. 14
Pyruvate Dehydrogenase complex (PDC)
Pyruvate Dehydrogenase complex (PDC):
• Critical step linking glycolysis to TCA
• Similar to aKGDH (Fig. 20.15)
• Huge complex;
• Many copies each subunit:
(Beef heart 30 E1, 60 E2, 6 E3, X)
Fig. 15
Regulation of PDC
PDC regulated mostly by phosphorylation:
• Both enzymes in complex
• PDC kinase add PO4 to ser on E1
• PDC phosphatase removes PO4
• PDC kinase:
•
•
inhibited by ADP, pyruvate
Activated by Ac CoA, NADH
Fig. 16
TCA cycle intermediates and anaplerotic paths
TCA cycle intermediates - biosynthesis precursors
• Liver ‘open cycle’ high efflux of intermediates:
• Specific transporters inner mitochondrial membrane
for pyruvate, citrate, a-KG, malate, ADP, ATP.
Fig. 17
GABA
Anaplerotic reactions
Anaplerotic reactions replenish 4-C needed to
regenerate oxaloacetate and keep TCA cycling:
• Pyruvate carboxylase
• Contains biotin
• Forms intermediate with CO2
• Requires ATP, Mg2+ (Fig. 8.12)
• Found in many tissues
Fig. 18
Amino acid degradation forms TCA cycle intermediates
Amino acid oxidation forms many TCA cycle
intermediates:
• Oxidation of
even-chain fatty acids and
ketone body not replenish
Fig. 19
Key concepts
• TCA cycle accounts for about 2/3 of ATP generated
from fuel oxidation
• Enyzmes are all located in mitochondrial
• Acetyl CoA is substrate for TCA cycle:
• Generates CO2, NADH, FAD(2H), GTP
• e- from NADH, FAD(2H) to electron-transport chain.
• Enzymes need many cofactors
• Intermediates of TCA cycle are used for
biosynthesis, replaced by anaplerotic (refilling)
reactions
• TCA cycle enzymes are carefully regulated
Nuclear-encoded proteins in mitochondria
Nuclear-encoded proteins enter
mitochondria via translocases:
• Proteins made on free ribosomes,
bound with chaperones
• N-terminal aa presequences
• TOM complex crosses outer
• TIM complex crosses inner
• Final processing
• Membrane proteins similar
Fig. 20
Review question
Succinyl dehydrogenase differs from other enzymes
in the TCA cycle in that it is the only enzyme that
displays which of the following characteristics?
a. It is embedded in the inner mitochondrial
membrane
b. It is inhibited by NADH
c. It contains bound FAD
d. It contains fe-S centers
e. It is regulated by a kinase