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Transcript
Copyright
COMMONWEALTH OF AUSTRALIA
Copyright Regulation
WARNING
This material has been reproduced and communicated to you by or
on behalf of the University of Sydney pursuant to Part VB of the
Copyright Act 1968 (the Act).
The material in this communication may be subject to copyright
under the Act. Any further reproduction or communication of this
material by you may be the subject of copyright protection under
the Act.
Do not remove this notice
Fatty acid oxidation
• Also called beta-oxidation
– Because the action occurs on the beta-carbon
atom
Fatty acid oxidation
• Requires tissues to have mitochondria
• Reciprocally regulated with glucose
oxidation
– Fatty acid oxidation inhibits glucose oxidation
• Consumes a lot of FAD, NAD, CoA
– Availability of cofactors is important
Transport of FA
Fatty acid binding protein
Albumin
Transport of FA
• FA needs to be transported from blood into tissues
• FA is carried in blood on albumin
– Several binding sites for FA
• There are specific transporters for FA
– CD36 moves to the cell surface whenever there is a
need to take up FA at a rapid rate
• FA is carried on FABP (fatty acid binding protein) in
cytoplasm
Trapping of FA
• FA is trapped by attaching it to CoA
• This also ‘activates’ the fatty acid (‘tags’ the FA)
• Requires quite a lot of energy,
– ATP is not converted into ADP, but AMP
Transport of FA: Mitochondria
FA-CoA is oxidized
Example: 16C FA-CoA
• 7 NADH & 7 FADH2 are produced…. NAD & FAD needed
• 8 acetyl CoA produced…. CoA needed
Cofactor Availability
• NAD, FAD and CoA
– All needed to keep FA oxidation going
• How are these carriers regenerated?
– CoA
• By entry of acetyl CoA into Krebs Cycle
– NAD/FAD
• By giving cargo to electron transport chain
Rate Limiting Enzymes
• The slowest enzyme in the metabolic
pathway determines the overall speed
– Rate-limiting step (RLS)
– Flux generating step
• Key points of regulation
Enzyme kinetics 
•
Vmax
•
Rate
½ Vmax
Km
S1
[substrate]
S2
At high [substrate],
minor changes in
[substrate] will not
affect the rate of
reaction
Doubling or
halving the [S] isn’t
even going to
affect the rate
Redfern Station Analogy
•
Imagine the station at peak hour with just
one barrier operating
– This gate will soon become ‘saturated’ with
people
– Increasing the number of people doesn’t
increase the rate
– It is the ‘rate limiting’ step
– The point which determines the overall rate at
which people get to Uni
Changing the Flux
•
There are 3 major ways to regulate this
(and metabolic!) pathways
– Change the intrinsic activity of the step
•
Make ticket-reading & gate-opening happen faster
– Make more gates open
•
•
•
Switch them from being ‘off’ to ‘on’
Or change the direction from ‘in’ to out
Or bring in a set of gates when you need them
– Make and destroy gates according to need
•
Seems crazy!
Properties of RLS
• Irreversible
– Need alternative enzymes to ‘go back’
– Not ‘equilibrium’ under physiological
conditions
– “Committed steps”
• Saturated with substrate
– Low Km or [S] >> Km
– Working at Vmax
RLS in FA ox?
•
•
•
•
Availability of fatty acids?
Cell membrane transport & Trapping?
Mitochondrial transport? Carnitine
Oxidation?
– Activity of enzymes
• Co-factor availability?
• Does it depend on the circumstances?
Glycolysis
•
•
•
•
•
•
Uses carbohydrate (glucose)
Wholly cytosolic
All cells of the body
No requirement for oxygen
Very, very fast
Very inefficient
Glucose
Glucose Uptake
P
hexokinase
glucose
blood
glucose
Using ATP
glucose
6-phosphate
cytoplasm
Uptake facilitated by Glucose transporters (GLUTs)
•GLUT-1 present in all cells all the time
•GLUT-4 muscle and adipose tissue (the insulin sensitive tissues)
•GLUT-2 liver and pancreas (blood glucose regulating tissues)
Early Glycolysis
Investment of energy giving a biphosphorylated symmetrical sugar
P
P
Phosphofructokinase
glucose
6-phosphate
PFK
fructose
6-phosphate
P
P
Using ATP
fructose
1,6-bisphosphate
Splitting to give two 3-carbon molecules
P
P
Two molecules of 3-carbon sugar phosphates
Return Phase
Remember two 3-carbon
molecules go down the pathway
P
Bring in phosphate
Oxidize with NAD
P
P
Super energy molecule!
Recoup some ATP
P
Recoup some ATP
pyruvate
Overview
• Total yield is 2 ATP per glucose
– And two pyruvate
– And two NADH
• Need to regenerate NAD
• Fate of the pyruvate
– Aerobic
– Anaerobic
Completing Glycolysis
• More ATP from oxidation of pyruvate
– Need to transport into mitochondria
– Oxidize with pyruvate dehydrogenase
• Need to reoxidise NADH
– To maintain the supply of NAD
– Shuttle systems available
• To send NADH electrons/hydrogens into matrix
– Lactate production
– In yeast, alcohol production
– Latter two keep everything cytosolic
Regulation
• Most points reversible
• Focus on three steps
– Hexokinase (G  G6P)
• Mainly feedback inhibition from G6P
– Phosphofructokinase (F6P  F6BP)
• Strongly affected by ATP/ADP levels
• But mainly via AMP levels
– Pyruvate kinase (last step)
• ATP/ADP important
Energy Charge
• Large changes in ATP not desirable
– Keep ATP at 5 mM
• Adenylate kinase
– Translates small change in ATP to large
relative change in AMP
– 2ADP  ATP + AMP
• Ratio of adenine nucleotide concentrations
often called ‘energy charge’
• Strong stimulation of PFK
Energy Charge
Integration of Catabolism
FA
BETA-OXIDATION
“CARNITINE”
FA-CoA
CD36
ac-CoA
ac-CoA
ac-CoA
FA-CoA
ac-CoA
ac-CoA
PDH
pyruvate
GLUT-4
glucose
citrate
GLYCOLYSIS
G6P
OAA
pyruvate
KREBS CYCLE
PHOSPHORYLASE
CO2
glycogen
CO2
IC
ICDH
2OG
OGDH
Krebs Cycle
• TCA cycle, Citric Acid cycle
• Substrate is acetyl CoA
– Fatty acid oxidation and/or glucose oxidation
• Overall strategy
–
–
–
–
Completely oxidize acetate carbons to CO2
Produce lots of NADH, FADH2, even an ATP
Perform the reaction on a carrier molecule
Regenerate the carrier
Regulation
• Krebs cycle activity is controlled early on
– At isocitrate dehydrogenase (ICDH)
– alpha-ketoglutarate dehydrogenase (OGDH)
• ICDH and OGDH are stimulated by rise in Ca2+
– Such as is found during exercise
• ICDH & OGDH are also sensitive to NAD levels
– Activity is dependent on availability of NAD
Important Features
• During the cycle
– 2 carbon atoms come in, 2 carbon atoms released
• Generates
– 3 NADH, 1 reduced FAD plus a GTP
– Each NADH gives 2.5 ATP in oxidative
phosphorylation
– Each FADH2 gives 1.5…
– So with the GTP, that’s about 10 ATP per acetate
• Oxaloacetate is not ‘net’ consumed in the cycle
– acts as carrier