Chapter 11: Glycolysis

1. Chapter 11: Glycolysis amylose sucrose lactose glucose fructose amylase -ose disaccharides -ase Active transport passive transport Low [Glc] High [Glc] Low [Glc] “Enjoy the chemical elegance of metabolism”

2. Chapter 11: Glycolysis Defined: Glucose is converted anaerobically to the three carbon acid pyruvate (only top half diagram) Net Reaction: Glucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O 1-4 = hexose (6C) stage: 2 ATP’s consumed. 5-10 = triose 2(3C) stage: 4 ATP’s produced. Net: 2 ATP’s. Max energy when pyruvate from glycolysis enters Citric Acid Cycle Makes reducing eqivalents NADH and QH2 (FADH2)

3. Glucose ATP Phosphorylation Hexokinase The Pathway ADP Glucose-6-phosphate Isomerization Glucose-6-phosphate isomerase Fructose-6-phosphate ATP Phosphorylation Phosphofructokinase-1 ADP Fructose-1,6-bisphosphate Cleavage Aldolase Dihydroxyacetone phosphate Isomerization Trios phosphate isomerase Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate NAD+ + Pi Oxidation and Phosphorylation Glyceraldehyde-3-phosphate dehydrogenase NAD+ + Pi NADH + H+ NADH + H+ 1,3-bisphosphoglycerate 1,3-bisphosphoglycerate ADP Substrate Level Phosphorylation ADP Phosphoglycerate kinase ATP ATP 3-phosphoglycerate 3-phosphoglycerate Phosphoglycerate mutase Rearrangement 2-phosphoglycerate 2-phosphoglycerate Enolase H2O Dehydration H2O phosphoenolpyruvate phosphoenolpyruvate ADP ADP Substrate Level Phosphorylation ATP Pyruvate kinase ATP pyruvate pyruvate

4. Glycolysis: Step 1, Hexokinase C-6 I-III IV Isozymes Different inhibition profiles Location, Km Control point Can’t leave the cell

5. Glycolysis: Step 2 Glucose 6-Phosphate Isomerase Aldose Opens the chain during the rxn CH2OH Ketose OH Stereospecific: uses -Glc; produces -D-fructose-6-phosphate

6. Step 3, Phosphofructokinase-1 First COMMITted step of glycolysis -anomer PFK-1 Phosphofructokinase-1 Other 6-C sugars can enter and convert to fru-6-phos fructose, mannose, etc. Metabolically irreversible rxn. It is an allosteric enzyme and a REGULATory CONTROL step for glycolysis (AMP and citrate).

7. Glycolysis: Steps 2 and 3 Opens the chain during the rxn PFK-1 CH2OH OH utilizes 100% -anomer Stereospecific: uses -Glc; produces 100% -D-fructose-6-phosphate 36% -fructose 64% -fructose

8. Glycolysis: Step 4, Aldolase Fig 11.5 Mech Rxn is near equilibrium, so not a control point Basic residue or metal withdraws an e- polarizing C2 carbonyl Basic residue removes a proton from the C4 hydroxyl group 1 2 3 4 5 6 Rapid depletion of 2 products in subsequent steps drives rxn

9. Step 5, Triose Phosphate Isomerase Ketose Aldose near equil. Only G3P, not DHAP, can be utilized in step 6 Now have 2 molecules G3P for step 6 Consumption in step 6 maintains steady state conc. of G3P Ketose-aldose conversion is diffusion controlled After conversion, C-1=C-6, C-2=C-5, C-3=C4 (see textbook)

10. Step 5, Triose Phosphate Isomerase Ketose Aldose near equil.

11. Glycolysis: Step 6 Glyceraldehyde 3-Phosphate Dehydrogenase from Pi Higher group transfer potential than ATP Oxidation has neg G, some energy conserved in acid anhydride linkage Oxidation is coupled to phosphorylation to conserve energy, instead of oxidation to free carbonic acid and energy loss

12. Step 7, Phosphoglycerate Kinase First ATP generating step Substrate level phosphorylation- Nucleotide diphosphate phosphorylated Donor is not a nucleotide Near equilibrium rxn. Reversibility is important for reverse step in glucose synthesis (gluconeogenesis). Really steps 6&7 couple oxidation to phosphorylation of ADP

13. Glycolysis: Step 8, Phosphoglycerate Mutase iPGM -cofactor independant dPGM -cofactor dependant

14. Step 9, Enolase phosphomonoester Enol-phosphate ester PEP: Very high P-group transferpotential

15. Glycolysis: Step 10, Pyruvate Kinase 2nd sub level phosphorylation 3rd metabolically irreverible rxn Reg allosteric and covalent modification

16. Glucose ATP #1 Phosphorylation Hexokinase ADP Glucose-6-phosphate Three Metabolically Irreversible Reactions Isomerization Glucose-6-phosphate isomerase Fructose-6-phosphate #3 ATP Phosphorylation Phosphofructokinase-1 ADP Fructose-1,6-bisphosphate Cleavage Aldolase Dihydroxyacetone phosphate Isomerization Trios phosphate isomerase Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate NAD+ + Pi NAD+ + Pi Oxidation and Phosphorylation Glyceraldehyde-3-phosphate dehydrogenase NADH + H+ NADH + H+ 1,3-bisphosphoglycerate 1,3-bisphosphoglycerate ADP Substrate Level Phosphorylation Phosphoglycerate kinase ADP ATP ATP Most are near equilibrium 3-phosphoglycerate 3-phosphoglycerate Phosphoglycerate mutase Rearrangement 2-phosphoglycerate 2-phosphoglycerate Enolase H2O Dehydration H2O phosphoenolpyruvate phosphoenolpyruvate ADP ADP #10 Substrate Level Phosphorylation ATP Pyruvate kinase ATP pyruvate pyruvate

17. Energetics of Glycolysis • Hexokinase • Phosphofructokinase 1 • 10. Pyruvate Kinase Enter the Le Chatelier Few large -G steps, irreversible, regulated * * * Most are near equilibrium and have G close to zero Steps 2, 4, 5, 6, 7, 8, 9

18. Physiological Regulation of Glycolysis Hormones Involved High blood [Glc], insulin released Low blood [Glc], glucagon released Insulin Independent Uptake Brain Liver Red Blood Cells Insulin Dependent Uptake Muscle Adipose

19. Regulation of Glycolysis by Cellular Import Hexose Transporters transport glucose into cells in an insulin dependent manner [Glucose] high in bloodsteam and low inside most cells- passive transport Exceptions Small intestine Kidney Which cell types are affected? High Insulin hormone and [Glc] stimulate increased rate of glucose intake Insulin binds receptor, GLUT4 hexose transporter able to bind cell surface

20. Enzymatic Regulation of Glycolysis Not moving forward, stop converting ATP Cellular rxns are converting ATP and ADP, make more ATP You’ve committed! Bi-phosphated furanoses, keep pathway moving CAC intermediates, slow down, there is already adequate supply of energy Regulation network samples the condition of the cytoplasm and applies Principles of Supply and Demand

21. Glycolysis: Hexokinase Isozymes Hexokinases (I-III) -regulated negatively by Glc-6-P -if later steps slow down, Glc-6P builds up I-III IV Isozymes Different inhibition profiles Location, Km Control point Glucokinase (IV) in Liver -regulated negatively by Fru-6-P -pulls glucose out of bloodstream until equil -liver can produce more Glc-6-P -converts Glucose to Glycogen storage Can’t leave the cell with negative charge

22. Regulation of Phosphofructokinase-1 Large oligomeric enzyme bacteria/mammals - tetramer yeast - octamer ATP - product of pathway - allosteric inhibitor AMP - allosteric activator - relieves inhibition by ATP Citrate - feedback inhibitor - regulates supply of pyruvate - links Glycolysis and CAC Fru-2,6-bisphosphate - strong activator - produced by PFK-2 when excess fru-6-phosphate - indirect means of substrate stimulation or feed forward activation

23. Regulation of Pyruvate Kinase + F 1,6 BP Inactivation by covalent modification -blood [Glc] drops, glucagon released -liver protein kinase A (PKA) turned on -PKA phosphorylates pyruvate kinase Allosteric (feed-forward) activation Fructose-1,6-bisphosphate -allosterically activates -produced in step three -links control steps together Allosteric inhibition by ATP -product of pathway and CAC Low blood [Glc] High blood [Glc]