iii. metabolism - gluconeogenesispeople.uleth.ca/~steven.mosimann/bchm3300/bchm3300_l9.pdf ·...
TRANSCRIPT
Biochemistry 3300 Slide 1
III. Metabolism
- Gluconeogenesis
Department of Chemistry and BiochemistryUniversity of Lethbridge
Biochemistry 3300
Carl & GertrudeCori
Biochemistry 3300 Slide 2
Carbohydrate Synthesis
Lactate, pyruvate and glycerolare the important 3C compoundsthat feed gluconeogenesis.
Glucogenic amino acids –are catabolized to pyruvate, andother citric acid cycle compoundsthat can enter gluconeogenesis.
Photosynthetic organisms usethe pathway to fix CO2.
Occurs in the liver of mammals !
Biochemistry 3300 Slide 4
The Cori Cycle
When a working muscle goes anaerobic,the generated lactate is excreted, thentransported to the liver via the bloodstream.
Liver:lactate is converted to glucose (gluconeogenesis).
Glucose (liver) is made available, through the bloodstream, to other tissuese.g. muscle.
Resting muscle:Glucose is still transported to the muscle to replenish glycogen levels.
Biochemistry 3300 Slide 6
Gluconeogenesis
Glycolysis and gluconeogenesisshare 7 of 10 steps
Reactions of hexokinase, PFK-1and pyruvate kinase are essentiallyirreversible in vivo.
These steps are bypassedin gluconeogenesis and requirenew, different enzymes.
The gluconeogenesis enzymereactions are also irreversiblein vivo.
Both are reciprocally regulated,cytosolic pathways. Metabolite flux is always in one direction.
Biochemistry 3300 Slide 7
Gluconeogenesis Reactions
Glucose 6-phosphatase and Fructose 1,6- bisphosphatase catalyze simplehydrolysis reactions. → strongly favorable
Biochemistry 3300 Slide 8
Gluconeogenesis Reactions
Conversion of pyruvate to PEPis complex.
Oxaloacetate is both a citric-acid cycle and a gluconeogenesismetabolite → occurs in mitochondria
Pyruvate is transported intothe mitochondrion to enter gluconeogenesis
Biochemistry 3300 Slide 9
Gluconeogenesis – Step 1
Two different pathways are possible !
Regulation:depends on the presence of
lactate in the cytosol.
The mitochondrion has no oxaloacetate transporter!
Note: pyruvate carboxylaserequires ATP hydrolysis & PEPcarboxykinase requires GTPhydrolysis
Difference:malate dehydrogenase
Biochemistry 3300 Slide 10
Gluconeogenesis – Step 1
Pyruvate carboxylase
1 ATP is used !C-C bond formed
Biotin – aka vitamin B7or vitamin H (another coenzyme)
Biochemistry 3300 Slide 11
Gluconeogenesis – Step 1
Biotin is covalently attached to thethe ε-amino group of lysine.
Reaction involves twodifferent active sites on thesame enzyme.
Site 1: Form carboxybiotin from HCO
3- and ATP
Site 2: Form oxaloacetate from pyruvate and released CO
2
Biochemistry 3300 Slide 12
Gluconeogenesis – Step 1
Active site 1Bicarbonate is convertedto CO
2 which reacts with
Biotin forming –carboxybiotin
Active site 2Carboxybiotin releases CO
2 which reacts with
pyruvate forming – oxaloacetate
Biochemistry 3300 Slide 13
Gluconeogenesis – Step 2
PEP carboxykinase
Oxaloacetate is converted toPEP using GTP as phosphoryldonor.
GTP is often used as a energysource in anabolism
Biochemistry 3300 Slide 14
Gluconeogenesis – Steps 1 & 2
Net cost for the reactions is 2 ATP (or 1 ATP + 1 GTP)
PEP carboxykinase reversibly exchanges high energy bonds (GTP for PEP)
∆G’o = 0.9 kJ/mol, but under cellular conditions ∆G’ = -25 kJ/mol
Biochemistry 3300 Slide 16
Pentose Phosphate Pathway
Alternative path for glucose oxidization
Electron acceptor is NADP+.
NADPH is needed for reductive biosynthesisAND
prevents oxidative damage to proteins at high levels (red blood cells, cornea)
Products: pentose phosphates +NADPH
Pentosephosphates
Biochemistry 3300 Slide 17
Pentose Phosphate Pathway – Oxidative Phase
Oxidations (dehydrogenases) have large negative free energy changes and are essentially irreversible.
Two NADPH are produced per G6P starting molecule - by the dehydrogenase reactions.
cyclicester
hydrolysis
oxidativedecarboxylation
isomerization
1 pentose phosphates is produced per G6P starting molecule
Biochemistry 3300 Slide 18
Pentose Phosphate Pathway – Oxidative Phase
Step 1:Cyclic ester product
Cyclic aldose sugar tocyclic sugar acid
Biochemistry 3300 Slide 19
Pentose Phosphate Pathway – Oxidative Phase
Step 2:Hydrolysis of cyclic ester
Cyclic sugar acidto linear sugar acid
Biochemistry 3300 Slide 20
Pentose Phosphate Pathway – Oxidative Phase
Step 3:Oxidative
decarboxylation
Acid hexose toketopentose
Biochemistry 3300 Slide 21
Pentose Phosphate Pathway – Oxidative Phase
Step 4:Isomerisation
Ketopentose toaldopentose
Biochemistry 3300 Slide 22
Pentose Phosphate Pathway – Nonoxidative Phase
??Choreographed
Dance??
Ribulose-5-phosphateis converted back intoGlucose-6-phosphate
Biochemistry 3300 Slide 23
Pentose Phosphate Pathway – Nonoxidative Phase
Overall:5C sugars are converted to 6C sugars.
G6P is regenerated from pentose phosphates to make more NADPH.
Enzymes are all cytosolicand unique to PP pathway
PP pathway shares intermediates withglycolysis/gluconeogenesis
Note: Epimerase actually convertsribulose 5-phosphate to xylulose5-phosphate
Biochemistry 3300 Slide 24
Pentose Phosphate Pathway- Nonoxidative Reactions
5 hexoses (6C) are madefrom 6 pentoses (5C)
Every reaction shown here is reversible !
Hexoses (blue) are fructose-6-phosphatePentoses (pink) are derived from ribulose-5-phosphate
Biochemistry 3300 Slide 25
Epimerase
Reaction utilizes an enediol intermediate similar tophosphopentose isomerase.
Epimerase reaction abstracts then adds a proton to C3,results in an inversed configuration on the carbon atom.
Ribulose 5-phosphate3-epimerase
Biochemistry 3300 Slide 26
Transketolase
Transfer of 2-carbon groupTPP-mediated - still bond-breaking but substrate is not an α-keto acid
Remember the Coenzyme ?
Biochemistry 3300 Slide 27
Thiamine DeficiencyThe ability of TTP’s thiazolium ring to add carbonyl groups and act as an “electron sink” makes it the coenzyme most utilized in α-keto acid decarboxylations.
Thiamin (vitamin B1) is neither synthesized nor stored in significant amounts byvertebrates. Deficiency in humans results in an ultimately fatal condition knownas beriberi.
Biochemistry 3300 Slide 28
Transaldolase
Transfer of 3-carbon unit;similarity to aldolase cleavage reaction in glycolysis.
Biochemistry 3300 Slide 29
Short Reminder – Class I Aldolase
Schiff's base mediatedC-C bond cleavage
Biochemistry 3300 Slide 30
Mechanistic Similarity !
Transketolase
Transaldolase
TPP stabilizes the two-carbon carbanion
Schiff's base stabilizes the three-carbon carbanion