Block 2 TCA Cycle Flashcards
What is the net reaction of acetyl CoA synthase?
Acetate + CoASH + ATP –> AcCoA + AMP + PPi
Where are the two types of AcCoA synthase found?
ACS1 - liver, cytosolic
ACS2 - heart & muscle, in mitochondrial matrix
How is the high-energy phosphate bond of GTP formed?
Substrate level phosphorylation catalyzed by succinate thiokinase
How many ATPs are produced per NADH and FADH2 in ETC? What’s the net yield per acetyl group oxidized?
2.5 ATP/NADH, 1.5/FADH2
Net yield: about 10 ATP/acetyl group (3 NADH, 1 FADH2, 1 GTP)
For the complete oxidation of glucose, where do the produced ATPs come from, and what is the overall yield?
Per pyruvate (2/glucose), 1 NADH from PDH (2.5), 3 NADH in TCA (7.5), 1 FADH in TCA (1.5), 1 GTP in TCA (1.0), glycolysis (2), 2 NADH from glycolysis (3 or 5). Total = 30-32.
What is citrate synthase (CS)?
1st enzyme TCA, AcCoA + OAA -> citrate (C). Irreversible, -7.7 kcal.
What inhibits citrate synthase? What reverses its effects?
Citrate; ATP citrate lyase (cytosol)
What is aconitase (A)?
2nd enzyme TCA, citrate (C3 OH) -> isocitrate (I) (C2 OH) with cis-aconitate intermediate; contains iron-sulfer cluster (Fe-cysteine, not heme); +1.5 kcal
What is isocitrate DH (ID)?
3rd TCA; decarboxylates isocitrate to alpha-ketoglutarate; irreversible, -5.3 kcal; also yields NADH
What is alpha-ketoglutarate DH (KD)?
4th TCA; ox decarboxylation of a-KG to succinyl CoA; -8 kcal
What are the required coenzymes for alpha-KG?
Thiamine pyrophosphate (TPP), lipoic acid (carb + protein), AcCoA, FAD, NAD+
How is the energy released from a-KG DH reaction conserved?
Stored in reduced NADH & thioester bond of succinyl CoA
What are the three enzymes that are part of the a-KG complex?
1) a-KG DH
2) Transsuccinylase
3) Lipoamide DH
What does a-KG DH do as part of the a-KG complex?
Carries weakly bound TPP and decarboxylates it
What does transsuccinylase do as part of the a-KG complex?
Transfers remaining 4 carbons of a-KG from TPP to AcCoA = succinyl CoA (reducing lipoic acid)
What does lipoamide DH do as part of the a-KG complex?
Transfers electrons from reduced lipoic acid to FAD then NAD+. Resulting NADH contains most of the free energy.
What is succinate thiokinase/succinyl CoA synthase (ST)?
5th TCA; converts sucCoA to succinate to trap high energy of sucCoA thioester bond in GTP
What is nucleoside diphosphate kinase?
Converts GTP + ADP to GDP + ATP
What is substrate level phosphorylation?
Formation of high energy phosphate bond where none existed previously, without use of molecular O2; -0.7 kcal
What is succinate DH (SD)?
6 TCA; oxidizes succinate to fumarate; contains Fe-S cluster; uses covalently attached (via His) FAD to accept 2 e-; imbedded in IMM; 0 kcal
What is fumarase (F)?
7 TCA; adds H2O to fumarate, to oxidize to malate; 0 kcal
What is fumarase deficiency/fumaric aciduria/Polygamist Down’s?
Children of cousin parents, neonatal polyhydramnios, encephalopathy, mental retardation, unusual facial features, brain malformations, seizures
What is malate DH (MD)?
8 TCA; oxidizes C-2 OH of malate to form OAA using NAD+ as e- acceptor; +7.1 kcal
What is the net energetics of the TCA cycle?
-13.1 kcal
What is the significance of the energy released from cleavage of AcCoA?
Makes entry into TCA cycle more thermodynamically favorable
What is the significance of the energy released from cleavage of succinyl CoA?
Provides energy for generation of GTP
From where does the energy of formation of AcCoA from pyruvate come?
Oxidative decarboxylation of pyruvate by PDH
In what other pathway can citrate be used?
Fatty acid synthesis
In what other pathway can a-KG be used?
Amino acid synthesis, and neurotransmitter synthesis (glutamate, GABA)
In what other pathway can succinyl CoA be used?
Heme synthesis (condensed with glycine to form delta-aminolevulinic acid, a heme precursor)
In what other pathway can malate be used?
Gluconeogenesis
In what other pathway can OAA be used?
Amino acid synthesis
How is gluconeogenesis supplied during a fast?
In liver, gluconeogenic precursos converted to malate, leaves mitos via specific transporter to enter cytosolic gluconeogenic pathway
What happens to citrate in the liver after a meal?
Converted to AcCoA and OAA by citrate lyase. AcCoA is precursor for FAs.
What is an anaplerotic reaction?
Reactions that supply 4-carbon intermediates to TCA cycle so it can keep running
What are the 4-C intermediates of the TCA cycle?
Succinate, fumarate, malate, OAA
What is the anaplerotic reaction supplying OAA?
Conversion of pyruvate and CO2 to OAA by pyruvate carboxylase (with biotin to form covalent intermediate with CO2)
What happens if OAA levels in the TCA cycle drop?
Rate of citrate synthesis decreases, increase in AcCoA, stimulates pyruvate carboxylase
What is pyruvate carboxylase?
Converts pyruvate + CO2 –> OAA; also converts alanine and lactate to glucose
How is OAA converted to phosphoenolpyruvate?
By phosphoenolpyruvate carboxykinase in the cytosol
What are the reagents in the anaplerotic reaction supplying succinyl CoA?
Isoleucine, valine, methionine
What is the anaplerotic pathway that supplies a-KG?
His, Pro, Gln, Arg -> glutamate a-KG by transaminases & glutamate DH
What is aspartate aminotransferase?
Converts Asp + a-KG OAA + Glu
What are the reagents the anaplerotic reaction supplying fumarate?
Tyrosine, phenylalanine
What drives the TCA cycle? What are the feedback mechanisms regulating it?
Rate of ATP use; phosphorylation state of ATP and reduction state of NAD+ or NADH/NAD+ ratio
What is the rate-limiting step of the TCA cycle?
a-KG DH because it has a low catalytic capacity under physiological conditions
What are the subunits of the pyruvate DH complex?
Catalytic: pyruvate dehydrogenase with TPP, dihydrolipoyl transacetylase with lipoamide, dihydrolipoyl DH/reductase with FAD.
Regulatory: PDH kinase, PDH phosphatase
What is PDH kinase?
Regulatory subunit of PDHC, P serine residue on PDH to inactivate it, is inhibited by ADP
What is PDH phosphatase?
Removes P to activate PDH
What are the activators and inhibitors of PDH?
Act: pyruvate, NAD+, insulin, ADP, Ca2+ (also ADH, a-adrenergic agonists)
Inh: AcCoA, NADH, ATP
What symptoms are displayed by children with PDHC deficiency?
Elevated serum lactate, pyruvate, alanine = chronic lactic acidosis; sever neurological defects, usually death
What are potential treatments for PDHC deficiency?
Ketogenic diet, minimizing carbohydrates; dichloroacetate to increase PDHC activity by inhibiting PDHC kinase; carnitine loading; thiamin
How might thiamin and carnitine loading help PDHC patients?
Thiamin for thiamin-deficient patients or patients with PDHC E1 subunit with abnormally high Km/low affinity for TPP; carnitine helps long-chain FA into mito, increasing ketone body formation by liver
What is the most prevalent defect in PDHC deficiency? How is the PDHC diagnosis made?
E1 component; diagnose by measuring PDHC in cultured skin fibroblasts
Why are PDHC E3 defects unique?
E3 gene shared by other a-keto acid DH like a-KG DH and branched chain AA like valine, leucine, isoleucine
What is beriberi? What causes its symptoms?
Thiamin deficiency -> neuro and cardio disorders. Severely malnourished alcoholics are also deficient. Symptoms: thiamin required for PDH, a-KG DH, and transketolase to transfer activated aldehyde units
In a person with beriberi, what happens after ingestion of glucose?
Blood pyruvate is especially high, but PDH and a-KG DH activity levels abnormally low = low glucose to nervous system
Why are mercury and arsenite dangerous?
Both have high affinity for neighboring sulfhydryl groups and inhibits dihydrolipoyl DH part of PDH = CNS pathology
How are beriberi and arsenic poisoning treated?
Sulfhydryl reagents with adjacent sulfhydryl groups to compete with dihydrolipoyl groups to bind metal ion; e.g. 2,3-dimercaptopropanol
