Glycolysis and Gluconeogenesis

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) Triose phosphate isomerase (TPI) • -> Isomerisation accelerated 1010-fold • -> Kcat/Km = 2 108 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