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Glycolysis and Gluconeogenesis

1. Energy –conversion pathway

2. Pathway tightly regulated

3. Synthesis of glucose from non-

CH procusors

4. Glycolysis and Gluconeogenesis

are reciprocally regulated

Glucose metabolism generates ATP -> powers muscle contraction

Glucose is generated by Dietary Carbohydrates

Starch + glycogen: main source of glucose

Mainly brocken down by α-amylase (cleaves α 1->4)

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Glycolysis is an Energy-Conversion Pathway in Many Organisms

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Glycolysis is an Energy-Conversion Pathway in Many Organisms

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Stage 1: Preparation of glucose by phosphorylation

-> Trapping of glucose in the cytosol

-> High-energy forms of glucose: destabilisation -> activation of glucose

Kinases: Phosphorylate substrates

-> Induced-fit mechanism of substrate recognition: closure of cleft

-> Shields active site from water

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Stage 1: Preparation of glucose by phosphorylation

Phosphoglucose isomerase

-> Conversion of aldose into ketose -> preparation for addition of second phosphate group

-> isomerase: open hemiacetal -> isomerisation -> close hemiketal

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Stage 1: Second phosphorylation

Phosphofructokinase -> control point of glycolysis -> allosteric enzyme

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Stage 2: Cleavage of C6 into 2x C3

Aldolase -> catalysis reverse aldol condensation

directly used in glycolysis

Not directly used in glycolysis

ketose aldoseIsomers

Reaction driven in GAP direction by removal of product through glycolysis8

Stage 2: Triose Phosphate Isomerase (TPI)

Triose phosphate isomerase (TPI)

-> Isomerisation accelerated 1010-fold

-> Kcat/Km = 2 108 M-1 s-1 -> kinetically perfect enzyme

-> suppresses an undesired side reaction

Reaction 100 times faster

TPI traps enediol intermediate -> prevents side reaction -> opens again when GAP formed

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Stage 3: Oxidation of C3 and ATP production -> Pay Off Phase

2 steps in one reaction:

1. Reaction -> thermodynamically favorable2. Reaction -> not favorable

ΔG°´= -50 kJ mol-1

ΔG°´= +50 kJ mol-1

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Stage 3: Mechanism of GAP dehydrogenase

Formation of thioester intermediate makes 2nd reaction (phosphorylation) possible !!

Transfer of a hydride ion (H-) to NAD+

Attack of the thioester by orthophosphate ion

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Formation of ATP in this manner -> Substrate-level phosphorylation

Stage 3: Formation of ATP

Rearrangement of phosphoryl group

Dehydration: formation of enol phosphate

Higher phosphoryl-transfer potential (Phosphoryl group traps molecule in unstable enol form)

Irreversible reaction -> ATP is profit!!!!!

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Summary of glycolysis

-> 10 reaction steps

-> 1 x C-6 (glucose) converted into 2x C-3 (pyruvate)

-> oxidation of glucose -> 2 NADH generated

-> 2 ATPs used + 4 ATPs generated -> pay off: 2 ATPs

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Glucose Metabolism Under Aerobic and Anaerobic Conditions

Final Electron-acceptor:

Aerobic -> O2

Anaerobic -> Pyruvate

Cytosol

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Why do we need to produce lactate or ethanol (yeast) anaerobic and not stop at pyruvate?

Gycolysis: Oxidation reaction generates NADH from NAD+

Under anaerobic conditions: reaction from Pyruvate to Lactate or Ethanol -> regenerate NAD+

Under aerobic conditions: regeneration of NAD+ happens in respiratory chain (mitochondria) -> via 2 different shuttles

-> Regeneration of NAD+

Entry points for other sugars into glycolysis

Uridine diphosphate galactose

Galactose toxic if transferase is missing

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Glycolysis is tightly regulated

• 2 major metabolic needs: ATP and Pyruvate (Acetyl-CoA)

• Enzymes catalysing irreversible reactions: sites of control (allostery)

• Hexokinase, phosphofructokinase, pyruvate kinase

• Allosteric control (ms), phosphorylation (s), transcriptional regulation (h)

Phosphofructokinase: the key enzyme in glycolysis control

• Inhibited by ATP (reversed by AMP)• Inhibited by low pH• Inhibited by citrate (Citric acid cycle)

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Regulation of glycolysis in the muscle

ATP inhibits all 3 enzymes Need for ATP (high AMP) activates PFK

-> ATP based regulation

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Regulation of glycolysis in the liver

Regulation by: -> ATP -> glucose level in blood -> need for building bocks for biosynthesis

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Regulation of glycolysis in the liver

Proteins responsible for uptake of glucose into the cell -> regulate blood glucose level

Uptake of glucose (tranporters) -> metabolism of glucose

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Regulation of blood glucose level in the liver

Cancer and exercise affect glycolysis in a similar way

Tumors -> enhanced uptake of glucose -> enhanced glycolysis

Hypoxia: O2 deficiencyTumor cells grow too fast -> not enough O2 for aerobic process -> unaerobic conditions (lactate)-> glycolysis primary source for ATP production

-> induction of blood vessel growth

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Synthesis of glucose from non-carbohydrate precursors:

-> gluconeogenesis

• Brain and blood cells depend on glucose -> 160g/day (mainly for the brain)

• Glucose in the blood: 20g, as glycogen: 190g

• Starvation > 1day other metabolites for energy!

-> Gluconeogenesis pathway:

• Takes place in liver (and kidneys)

• Important to maintain blood glucose level

• Major precursors: glycerol, amino acids, lactic acid

• Specific enzymes in addition to glycolysis

(for the irreversible steps in glycosis) 23

Synthesis of glucose from non-carbohydrate precursors: -> gluconeogenesis

Triacylglycerols (Lipids) taken up by diet

-> brocken down to fatty acids and glycerol

cannot by converted to glucose

glucose

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Glycolysis <-> gluconeogenesis

Gluconeogenesis is not the reversal of glycolysis !!!

Glycolysis: in the cytosol

Gluconeogenesis: major part in cytosol-> 1st step in mitochondria -> shuttle

Reverse reaction of glycolysis thermodynamically not favorable !!!

Biotin: prosthetic group -> carrier for CO2

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Synthesis of glucose from non-carbohydrate precursors: -> gluconeogenesis

Pyruvate (end product of glycolysis) -> under aerobic conditions -> shuttle into Mitochondria -> converted into acetyl-CoA -> citric acid cycle

Gluconeogenesis -> start with pyruvate in mitochondria

1st Step: convertion to oxaloacetate

-> malate/oxaloacetate shuttleglycolysis

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Synthesis of glucose from non-carbohydrate precursors: -> gluconeogenesis

Free glucose is important control point -> pathway ends mostly with glucose-6-P -> finished just if glucose is needed (in blood) -> advantage of stopping at glucose-6-P -> trapped in the cell (cannot shuttle outside)

Last step of gluconeogenesis: in ER lumen -> glucose shuttled back to cytosol -> leaves cell

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Synthesis of other saccharides through gluconeogenesis

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Reciprocal regulation of glycolysis & gluconeogenesis

• Pathways not active at same time

• Regulated by products of reaction and precursors (allostery)

• Regulated by hormones: glucagon & insulin, through F-2,6-BP

• Regulated at the transcriptional level of genes

In the liver: aim is to maintain blood glucose level

glucagoninsulin

transcription

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Balance between glycolysis and gluconeogenesis in the liver -> sensitive to blood glucose concentration

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Regulated by a bifunctional enzyme: PFK2/FBPase2-> formed by PFK2-> hydrolysed (dephosphorylated) by FBPase2

Phosphofructokinase 2

Fructose bisphophatase 2

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Balance between glycolysis and gluconeogenesis in the liver -> sensitive to blood glucose concentration

Low blood-glucose level -> glucagon-> low level of F-2,6-BP

High blood-glucose level -> insulin-> high level of F-2,6-BP

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Pathway Integration during a sprint

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