III. Metabolism - Gluconeogenesispeople.uleth.ca/~steven.mosimann/bchm3300/Bchm3300_L9.pdf · Biochemistry 3300 Slide 1 III. Metabolism - Gluconeogenesis Department of Chemistry and

Biochemistry 3300 Slide 1

III. Metabolism

- Gluconeogenesis

Department of Chemistry and BiochemistryUniversity of Lethbridge

Biochemistry 3300

Carl & GertrudeCori


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 !


Glucogenic Amino Acids ?


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.


Glycolysis Again


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.


Gluconeogenesis Reactions

Glucose 6-phosphatase and Fructose 1,6- bisphosphatase catalyze simplehydrolysis reactions. → strongly favorable


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


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



Pyruvate carboxylase

1 ATP is used !C-C bond formed

Biotin – aka vitamin B7or vitamin H (another coenzyme)



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



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



PEP carboxykinase

Oxaloacetate is converted toPEP using GTP as phosphoryldonor.

GTP is often used as a energysource in anabolism


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


Summary


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


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



Step 1:Cyclic ester product

Cyclic aldose sugar tocyclic sugar acid



Step 2:Hydrolysis of cyclic ester

Cyclic sugar acidto linear sugar acid



Step 3:Oxidative

decarboxylation

Acid hexose toketopentose



Step 4:Isomerisation

Ketopentose toaldopentose


Pentose Phosphate Pathway – Nonoxidative Phase

??Choreographed

Dance??

Ribulose-5-phosphateis converted back intoGlucose-6-phosphate


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


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


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


Transketolase

Transfer of 2-carbon groupTPP-mediated - still bond-breaking but substrate is not an α-keto acid

Remember the Coenzyme ?


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.


Transaldolase

Transfer of 3-carbon unit;similarity to aldolase cleavage reaction in glycolysis.


Short Reminder – Class I Aldolase

Schiff's base mediatedC-C bond cleavage


Mechanistic Similarity !

Transketolase

Transaldolase

TPP stabilizes the two-carbon carbanion

Schiff's base stabilizes the three-carbon carbanion


Regulation

Excess NADPH

Glucose 6-phosphate dehydrogenaseis allosterically inhibited by NADPH.

ie. Feedback inhibition of committedstep of pentose phosphate pathway

III. Metabolism - Gluconeogenesispeople.uleth.ca/~steven.mosimann/bchm3300/Bchm3300_L9.pdf · Biochemistry 3300 Slide 1 III. Metabolism - Gluconeogenesis Department of Chemistry and

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