1. 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

2. Glucose is generated by Dietary Carbohydrates
Starch + glycogen: main source of glucose
Mainly brocken down by α-amylase (cleaves α 1->4)

3. Glycolysis is an Energy-Conversion Pathway in Many Organisms

4. Glycolysis is an Energy-Conversion Pathway in Many Organisms

5. 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

6. 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

7. Stage 1: Second phosphorylation
Phosphofructokinase -> control point of glycolysis
-> allosteric enzyme

8. Stage 2: Cleavage of C6 into 2x C3
Not directly used in glycolysis
directly used in glycolysis
Aldolase -> catalysis reverse aldol condensation
ketose
aldose
Isomers
Reaction driven in GAP direction by removal of product through glycolysis

9. Stage 2: Triose Phosphate Isomerase (TPI)
-> Isomerisation accelerated 1010-fold
-> Kcat/Km = M-1 s-1 -> kinetically perfect enzyme
-> suppresses an undesired side reaction
TPI traps enediol intermediate -> prevents side reaction -> opens again when GAP formed
Reaction 100 times faster

10. Stage 3: Oxidation of C3 and ATP production -> Pay Off Phase
2 steps in one reaction:
ΔG°´= -50 kJ mol-1
ΔG°´= +50 kJ mol-1
Reaction -> thermodynamically favorable
Reaction -> not favorable

11. Stage 3: Mechanism of GAP dehydrogenase
Transfer of a hydride ion (H-) to NAD+
Formation of thioester intermediate makes 2nd reaction (phosphorylation) possible !!
Attack of the thioester by orthophosphate ion

12. Stage 3: Formation of ATP
Formation of ATP in this manner -> Substrate-level phosphorylation
Rearrangement of phosphoryl group
Irreversible reaction -> ATP is profit!!!!!
Dehydration: formation of enol phosphate
Higher phosphoryl-transfer potential (Phosphoryl group traps molecule in unstable enol form)

13. 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

14. Glucose Metabolism Under Aerobic and Anaerobic Conditions
Final Electron-acceptor:
Aerobic -> O2
Anaerobic -> Pyruvate
Cytosol

15. Why do we need to produce lactate or ethanol (yeast) anaerobic and not stop at pyruvate?
-> Regeneration of NAD+
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

16. Entry points for other sugars into glycolysis
Uridine diphosphate galactose
Galactose toxic if transferase is missing

17. 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)

18. Regulation of glycolysis in the muscle
-> ATP based regulation
ATP inhibits all 3 enzymes
Need for ATP (high AMP) activates PFK

19. Regulation of glycolysis in the liver
Regulation by: -> ATP
-> glucose level in blood
-> need for building bocks for biosynthesis

20. 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

21. Regulation of blood glucose level in the liver

22. Cancer and exercise affect glycolysis in a similar way
Tumors -> enhanced uptake of glucose -> enhanced glycolysis
Hypoxia: O2 deficiency
Tumor cells grow too fast -> not enough O2 for aerobic process -> unaerobic conditions (lactate)-> glycolysis primary source for ATP production
-> induction of blood vessel growth

23. 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)

24. 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

25. Glycolysis <-> gluconeogenesis
Gluconeogenesis is not the reversal of glycolysis !!!
Glycolysis: in the cytosol
Gluconeogenesis: major part in cytosol
-> 1st step in mitochondria -> shuttle
Biotin: prosthetic group -> carrier for CO2
Reverse reaction of glycolysis thermodynamically not favorable !!!

26. 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 shuttle
glycolysis

27. 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

28. Synthesis of other saccharides through gluconeogenesis

30. 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
glucagon
insulin
transcription
In the liver: aim is to maintain blood glucose level

31. - Balance between glycolysis and gluconeogenesis in the liver
-> sensitive to blood glucose concentration
Regulated by a bifunctional enzyme: PFK2/FBPase2
-> formed by PFK2
-> hydrolysed (dephosphorylated) by FBPase2 -
Fructose bisphophatase 2
Phosphofructokinase 2

32. Balance between glycolysis and gluconeogenesis in the liver
-> sensitive to blood glucose concentration
High blood-glucose level -> insulin-> high level of F-2,6-BP
Low blood-glucose level -> glucagon-> low level of F-2,6-BP

33. Pathway Integration during a sprint