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Glycolysis
• Glucose → pyruvate (+ ATP, NADH)
• Preparatory phase + Payoff phase
• Enzymes
– Highly regulated (eg. PFK-1 inhibited by ATP)
– Form multi-enzyme complexes
• Pass products/substrates along: efficiency
Overall balance sheet
Glucose + 2NAD+ + 2ADP + 2Pi →
2 pyruvate + 2NADH + 2H+ + 2ATP +
2H2O
Fermentation pathways
Alternate fate of pyruvate
• “Fermentation”: carbohydrate
metabolism that generates
ATP but doesn’t change
oxidation state (no O2 used, no
net change in NAD+/NADH)
• Fermentation of pyruvate to
lactate
– Cells with no mitochondria
(erythrocytes)
– anerobic conditions
– Regeneration of 2 NAD+ to
sustain operation of
glycolysis
– No net change in oxidation
state (glucose vs lactate)
• Lactate is recycled to glucose
(post-exercise)
Fermentation pathways
• Fermentation of
pyruvate to EtOH
– Yeast and
microorganisms
– No net oxidation
(glucose to ethanol)
– EtOH and CO2
generated
Aerobic respiration of glucose (etc)
• Glycolysis:
– Start with glucose (6 carbon)
– Generate some ATP, some NADH, pyruvate (2 x 3 carbon)
• TCA cycle
– Start with pyruvate
– Generate acetate
– Generate CO2 and reduced NADH and FADH2
• Electron transport
– Start with NADH/FADH2
– Generate electrochemical H+ gradient
• Oxidative phosphorylation
– Start with H+ gradient and O2 (and ADP + Pi)
– Generate ATP and H2O
Aerobic respiration
• Stage 1:
– Acetyl CoA production
• Some ATP and reduced electron carriers (NADH)
• Glycolysis (for glucose), pre-TCA
• Stage 2:
– Acetyl CoA oxidation
• Some ATP, lots of reduced e- carriers (NADH/FADH2)
• TCA cycle/Krebs cycle/Citric acid cycle
• Stage 3:
– Electron transfer and oxidative phosphorylation
• Generate and use H+ electrochemical gradient
• Use of reduced e- to generate ATP
Fate of pyruvate under aerobic
conditions: TCA cycle (Ch. 16)
• Oxidation of pyruvate in ‘preTCA cycle’
– Generation of acetyl CoA (2
carbons)
– CO2
– NADH
• Acetyl CoA → TCA cycle
– Generation of ATP, NADH
Pre-TCA cycle
• Pyruvate acetyl CoA
– Via ‘pyruvate dehydrogenase complex’
• 3 enzymes
• 5 coenzymes
– ~irreversible
– 3 steps
• Decarboxylation
• Oxidation
• Transfer of acetyl groups to CoA
• Mitochondria
– Transport of pyruvate
• Coenzymes involved (vitamins)
– Catalytic role
– Thiamin pyrophosphate (TPP)
• Thiamin
• decarboxylation
– Lipoic acid
• 2 thiols disulfide formation
• E- carrier and acyl carrier
– FAD
• Riboflavin
• e- carrier
– Stoichiometric role
– CoA
• Pantothenic acid
• Thioester formation acyl carrier
– NAD+
• Niacin
• e- carrier
Pre-TCA cycle
Pre-TCA cycle
• Enzymes involved pyruvate dehydrogenase
complex
– multiprotein complex
– Pyruvate dehydrogenase (24) (E1)
• Bound TPP
– Dihydrolipoyl transacetylase (60) (E2)
• Bound lipoic acid
– Dihydrolipoyl dehydrogenase (12) (E3)
• Bound FAD
• 2 regulatory proteins
– Kinase and phosphatase
Pre-TCA cycle
• Step 1:
– Catalyzed by
pyruvate
dehydrogenase
• Decarboxylation
using TPP
• C1 is released
• C2, C3 attached to
TPP as hydroxyethyl
Pre-TCA
• Step 2
– Hydroxyethyl TPP is oxidized to form acetyl linkedlipoamide
– Lipoamide (S-S) is reduced in process
– Catalyzed by pyruvate dehydrogenase (E1)
• Step 3
– Acetyl group is transferred to CoA
– Oxidation energy (step 2) drives formation of
thioester (acetyl CoA)
– Catalyzed by dihydrolipoyl transacetylase (E2)
• Step 4
– Dihydrolipoamide is oxidized/regenerated to
lipoamide
– 2 e- transfer to FAD, then to NAD+
Overall….
• Pyruvate acetyl CoA
– Via ‘pyruvate dehydrogenase
complex’
– 4 step process
• Decarboxylation of pyruvate
and link to TPP
• Oxidation of hydroxyethyl TPP
and reduction/acetylation of
lipoamide
• Transfer of acetyl group to
CoA
• Oxidation of lipoamide via
FAD (and e- transfer to NAD+)
Overall….
• Pyruvate acetyl CoA
– Via ‘pyruvate dehydrogenase
complex’
– 4 step process
• Decarboxylation of pyruvate
and link to TPP
• Oxidation of hydroxyethyl TPP
and reduction/acetylation of
lipoamide
• Transfer of acetyl group to
CoA
• Oxidation of lipoamide via
FAD (and e- transfer to NAD+)
Pre-TCA
• Substrate channeling
– Multi enzyme complex
–  rxn rate
• Facilitated by E2
– ‘swinging’ lipoamide
– accept e- and acetyl from
E1 and transfer to E3
• Pathology: mutations in
complex/thiamin
deficiency
Regulation of pre-TCA
• PDH complex
– Inhibited by
• Acetyl CoA, ATP, NADH,
fatty acids
– Activated by
• CoA, AMP, NAD+
– Phosphorylation
• Serine in E1
phosphorylated by
kinase
– Inactive E1
– Kinase activated by
ATP, NADH, acetyl
CoA…
• Regulatory phosphatase
hydrolyzes the
phosphoryl
– Activates E1
– Ca2+ and insulin
stimulate
TCA cycle
• Aerobic process
– “Generates” energy
– Occurs in mitochondria
– 8 step process
• 4 are oxidations
• Energy ‘conserved’ in
formation of NADH and
FADH2
– Regenerated via
oxidative
phosphorylation
– Acetyl group → 2 CO2
• Not the C from the
acetyl group
– Oxaloacetate required in
‘catalytic’ amounts
– Some intermediates
• Other biological
purposes
• Step 1: condensation
of oxaloacetate with
acetyl CoA citrate
• Via citrate synthase
TCA cycle
– Conformational change
upon binding
– Oxaloacetate binds 1st
• Conf change to create
acetyl CoA site
• Citrate synthase
– Conformational changes
upon binding of
oxaloacetate
unbound
bound
TCA cycle
• Mechanism of citrate
synthase
• 2 His and 1 Asp
• 2 reactions
– 1st rxn (condensation)
• 2 steps
• Highly unfavorable
because of low
oxaloacetate
– 2nd rxn (hydrolysis)
• Highly favorable because
of thioester cleavage
• Drives 1st rxn forward
• CoA is recycled back to
the pre TCA cycle