Glycolysis & TCA Cycle Flashcards
Metabolism
chemical conversions in biological systems
- series of enzyme catalysed reactions with net thermodynamic favorability
Anabolism
Synthesis of macromolecules from precursor molecules
Catabolism
Breakdown of nutrients into ‘waste’
Main types of Metabolic Reactions
- hydrolysis/dehydration
- oxidation/reduction
- isomerisation
- C-C cleavage
- group transfer
Glycolysis
energy producing pathway also providing synthesis precursors
- first step in oxidation of glucose to carbon dioxide
- can occur with or without oxygen
Net Glycolysis Reaction
glucose + 2Pi + 2ADP + 2NAD+ –> 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H20
TCA Cycle
final common pathway for oxidation of all fuel molecules / can also give biosynthesis intermediates
2 Phases of Glycolysis
- Preparatory phase
- phosphorylation of glucose using 2 ATP
- cleavage to 2x3C sugars - Payoff phase
- oxidation of 3C sugars to product ATP
- each reaction happens twice per glucose molecule
Step 1 of Glycolysis
Phosphorylation of Glucose - C6
- Glucose 6 Phosphate formed
- hexokinase catalyses this
- activates glucose; some energy from ATP hydrolysis conserved in the molecule
- keeps it in the cell (no transporter available for it)
Step 2 of Glycolysis
Isomeration
- fructose 6 phosphate formed
- aldose to ketose sugar
- carbonyl group moved from C1 to C2
Step 3 of Glycolysis
Phosphorylation
- fructose 1,6 bisphosphate
- group transfer reaction of phosphate onto C1: both C phosphorylated ensures that both 3C sugars made will have phosphate groups
- phosphofructokinase catalyses this
Step 4 of Glycolysis
Carbon - Carbon cleavage
- C2 carbonyl facilitates C-C bond cleavage at correct position
- dihydroxyacetone phosphate and glyceraldehyde 3-phosphate formed
- dihydroxyacetone phosphate isomerised to glyceraldehyde 3-phosphate via intramolecular redox reaction (H transfer from C1 to C2), this is simply an isomerisation
Step 5 of Glycolysis
Oxidation by NAD+ and phosphorylation
- 1,3 bisphosphoglycerate
- uses inorganic phosphate
- phosphorylation coupled to glyceraldehyde 3-phosphate by a thioester intermediate
- energy from this oxidation trapped in 1,3 BPG to later power ATP production
Step 6 of Glycolysis
ATP Production
- net yield of ATP here is 0
- energy rich / high phosphoryl transfer power
- 3-phosphoglycerate formed
- phosphate from position 1 transferred to ADP
- group transfer reaction
Step 7 of Glycolysis
Phosphate Moved from 3 position to 2 position
- needed for final steps
- 2-phosphoglycerate formed
- phosphoglycerate mutase catalyses this
Step 8 of Glycolysis
Dehydration
- water removed to give phosphoenolpyruvate
- activating phosphate group for transfer to ADP
Step 9 of Glycolysis
ATP Production
- pyruvate formed
- unstable enol form stabilised
- high phosphoryl transfer potential arises from enol-ketone conversion driving force
- net yield of 2 ATP
- ATP made due to substrate level phosphorylation
Substrate Level Phosphorylation
Steps 6,9 of glycolysis
- mechanism of ATP synthesis in glycolysis
- both 1,3 BPG and phosphoenol pyruvate have higher phosphoryl transfer power than ATP
- they’re also unstable so it is favorable to transfer a phosphate to give a stable molecule
Aerobic Conditions
- glucose fully oxidised to carbon dioxide using coenzyme A and the TCA cycle
Anaerobic Conditions
- NAD+ not regenerated via oxidative phosphorylation so additional reactions are needed to regenerate them to continue glycolysis
- oxidised to ethanol or lactate
- buildup of these can be toxic however so limit glycolysis rate
Allosteric Regulation of Glycolysis
- done at key/irreversible points in the pathway
- hexokinase
- pyruvate kinase
- phosphofructokinase: key control point as this is the first commited step of glycolysis
- downregulated by ATP increases
Glycolysis in the Muscle vs. Liver (PFK 1 regulation)
Different isozymes of PFK
muscle:
- low ph = inhibition
- lactate production slows glycolysis down to prevent damage
- high ATP decreases affinity for substrate (energy charge ratio regulates enzyme)
liver: enzyme also controlled by concentrations of biosynthetic intermediates
- PFK inhibited by citrate
- fructose 2,6 BP activates PFK
Reversible Phosphorylation
- liver: pyruvate kinase
- hormone triggered phosphorylation (glucagon)
- phosphorylation by cyclic AMP dependent protein kinase makes enzyme less active
- low glucose levels = phosphorylation = glycolysis slowed to conserve glucose
- this prevents the liver from using up all the glucose
TCA Cycle
- complete oxidation of glucose to produce reduced electron carriers that feed into the ETC
- final common pathway of all fuel molecule oxidation
Pyruvate Dehydrogenase
- links glycolysis to TCA cycle
- oxidative decarboxylation of pyruvate to acetyl CoA in mitochondrial membrane
- large enzyme complex made of 3 enzymes and 5 cofactors
Reaction Mechanism of PDH
- decarboxylation
- oxidation
- transfer to CoA
Coenzyme A
- activated carrier of acyl groups
- contains reactive thiol group that reacts with carboxylic acid to form a thioester
- derives from vitamin B5
- thioester hydrolysis has large negative free energy so acetyl is readily transferred to other molecules
- CoA addition activates acetyl group
Net Reaction of TCA Cycle
acetyl CoA + 3NAD + FAD + ADP/GDP + Pi + 2H2O = 2CO2 + 3NADH + FADH2 + ATP/GTP + CoA + 2H+
- essentially an oxidation of acetyl CoA to carbon dioxide and reduction of electron carriers
- ATP synthesis is 2.5 ATP per NADH and 1.5 ATP per FADH2
Steps in TCA Cycle
- 2 carbon acetyl-CoA condensation (aldol condenstoin with hydrolysis) with oxaloacetate to form citrate (6C)
- isomerisation to isocitrate (reposition hydroxyl group to set up decarboxylation)
- oxidative decarboxylation to form a-ketoglutarate (5C)
- oxidative decarboxylation and CoA addition to form succinyl-CoA (4C)
- substrate level phosphorylation to succinate (forms GTP and uses thioester bond hydrolysis to drive synthesis)
- oxidation to form fumarate (FADH reduced because energy change of reaction not high enough to reduce NAD)
- hydration to for malate (water added across double bond)
- oxidation to form oxaloacetate (oxidation of hydroxyl to carbonyl) ; ie. regeneration of starting compound
ATP Generation from TCA Cycle
- substrate level phosphorylation of succinyl CoA to succinate
- oxidative phosphorylation using reduced electron carriers in ETC
NAD (nicotinamide adenine dinucleotide)
- nicotinamide ring
- synthesised from vit. B3
1. addition of 2H and 2e
2. removal of one proton - forms NADH
FAD (flavin adenine dinucleotide)
- flavin ring
- synthesised from vit. B2
1. addition of 2H and 2e at one time
Biosynthesis and Anabolism in TCA Cycle
- also produces building blocks for synthesis of biomolecules
- citrate gives fatty acids/sterols
- a-keto glutarate gives amino acids
- succinyl-CoA gives porphyrins
- oxaloacetate gives purines/pyrimidines and amino acids
Production of Oxaloacetate to replenish cycle
- carboxylation of pyruvate
- anaplerotic reaction
- enzyme: pyruvate carboxylase (biotin ring)
- biotin attached via amide linkage to lysine
- biotin used to attach bicarbonate that is then transferred to pyruvate
- uses ATP for energy
Allosteric Regulation of TCA cycle
- response to ATP levels in cell (energy charge)
1. pyruvate dehydrogenase (inhibited by ATP/NADH/acetyl CoA)
2. isocitrate dehydrogenase (inhibited by ATP/NADH)
3. a-keto glutarate dehydrogenase (inhibited by ATP/NADH/succinyl CoA)
Regulation of Pyruvate Dehydrogenase
- allosteric regulation and reversible phosphorylation
- phosphorylation of serine by PDH kinase on E1 subunit inactivates
- kinase is activated by ATP, acetyl CoA, NADH
- dephosphorylation of serine by PDH phosphatase on E1 subunit activated
eg. muscle phosphatase is activated by calcium ions. this signals that ATP production is needed and stimulates the TCA cycle
