1. GlycolysisGluconeogenesis

2. Glycolysis - Overview One of best characterized pathways Characterized in the first half of 20th century Glucose --> 2 pyruvates + energy Strategy add phosphoryl groups to glucose convert phosphorylated intermediates into compounds with high phosphate group-transfer potentials couple the subsequent hydrolysis of reactive substances to ATP synthesis Glucose + 2NAD + + 2 ADP + 2P i --> 2NADH + 2 pyruvates + 2ATP + 2H 2 O + 4H +

3. Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway Essentially all cells carry out glycolysis Ten reactions - similar in most cells - but rates differ Two phases: –First phase converts glucose to two G-3-P –Second phase produces two pyruvates Products are pyruvate, ATP and NADH NADH must be recycled Three possible fates for pyruvate

4. Glycolysis

6. Fate of pyruvate Decarboxylation to acetaldehyde Reduction to ethanol Reduction to lactate Mitochondrial oxidation 1 NADH --> ~3 ATP

7. Enzymes of glycolysis Catalyzed reactions and properties

9. Glucose Glucose-6-phosphate Fructose-6- phosphate Fructose-1,6- biphosphate Glyceraldehyde-3- phosphate Hexokinase, glucokinase Phosphoglucoisomerase Phosphofructokinase Aldolase Triose phosphate isomerase Dihydroxyacetone phosphate

10. First Phase of Glycolysis The first reaction - phosphorylation of glucose Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous

11. Hexokinase 1st step in glycolysis;  G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell K m for glucose is 0.1 mM; cell has 4 mM glucose So hexokinase is normally active! Glucokinase (K m glucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P -

13. Hexokinase First step in glycolysis Large negative deltaG Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P Corresponding reverse reaction (Gluconeogenesis) is catalyzed by a different enzyme (glucose-6- phosphatase) Is it the committed step in glycolysis ?

14. Glucose Fructose-6-P Glucose-6-P Glyceraldehyde-3-P Pyruvate ATP GlycogenRibose-5-P + NADPH Nucleic acid synthesis Reducing power Glucose-6-P dehydrogenase

15. Rx 2: Phosphoglucoisomerase Glucose-6-P to Fructose-6-P

17. Rx 3: Phosphofructokinase PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, neg delta G - means PFK is highly regulated ATP inhibits, AMP reverses inhibition Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high

20. Glycolysis - Second Phase Metabolic energy produces 4 ATP Net ATP yield for glycolysis is two ATP Second phase involves two very high energy phosphate intermediates. –1,3 BPG –Phosphoenolpyruvate

21. Glyceraldehyde-3- phosphate 1,3-biphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate phosphoenolpyruvate pyruvate Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase

22. Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis Large, negative  G - regulation! Allosterically activated by AMP, F-1,6-bisP Allosterically inhibited by ATP and acetyl- CoA

23. The Fate of NADH and Pyruvate Aerobic or anaerobic?? NADH is energy - two possible fates: –If O 2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation –In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD + for more glycolysis

24. The Fate of NADH and Py Aerobic or anaerobic?? Pyruvate is also energy - two possible fates: –aerobic: citric acid cycle –anaerobic: LDH makes lactate

25. The elegant evidence of regulation! Standard state  G values are scattered: + and -  G in cells is revealing: Most values near zero 3 of 10 reactions have large, negative  G Large negative  G reactions are sites of regulation! Energetics of Glycolysis

26. Gluconeogenesis Synthesis of "new glucose" from common metabolites Humans consume 160 g of glucose per day 75% of that is in the brain Body fluids contain only 20 g of glucose Glycogen stores yield 180-200 g of glucose So the body must be able to make its own glucose

27. Comparison of glycolysis and gluconeogenesis pathways

28. Substrates for Gluconeogenesis Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized Fatty acids cannot! Most fatty acids yield only acetyl-CoA Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars

29. Gluconeogenesis I Occurs mainly in liver and kidneys Not the mere reversal of glycolysis for 2 reasons: –Energetics must change to make gluconeogenesis favorable (delta G of glycolysis = -74 kJ/mol –Reciprocal regulation must turn one on and the other off - this requires something new!

30. Energetics of Glycolysis The elegant evidence of regulation!  G in cells is revealing: Most values near zero 3 of 10 reactions have large, negative  G Large negative  G reactions are sites of regulation! Reactions 1, 3 and 10 should be different to go into opposite direction

31. Gluconeogenesis II Something Borrowed, Something New Seven steps of glycolysis are retained: –Steps 2 and 4-9 Three steps are replaced: –Steps 1, 3, and 10 (the regulated steps!) The new reactions provide for a spontaneous pathway (  G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation