The biosynthesis of cell constituents II: glycolysis: steps 1-5

1. The biosynthesis of cell constituents II: glycolysis: steps 1-5 • Objectives • How cells make ATP: energy-releasing pathways • Food molecules are broken down in three stages • Carbohydrates and their principal biological roles • Glycolysis is a central ATP-producing pathway: define glycolysis and explain its role in the generation of metabolic energy Carbohydrates 1. Structure and nomenclature of carbohydrates 2. Give examples of complex carbohydrates 3. Where are glycoproteins used? Refer to chapters 11 & 16, Stryer 5e Lecture 17, Michael Schweizer

3. Diagram of the stages of cellular metabolism that lead from food to waste products in animal cells

4. Diagram of the stages of cellular metabolism that lead from food to waste products in animal cells

5. Glycolysis Glycolytic pathway Embden-Meyerhof pathway Glycolysis: glyk sweet; lysis, splitting Glycolysis takes place in the cytosol of cells.

6. Otto Meyerhof Karl Lohmann discovered ATP in 1929 http://sun0.mpimf-heidelberg.mpg.de/History/Meyerhof.html Gerty Theresa Cori & Carl Ferdinand Cori

7. Scientists Who Made Important Contributions to the Discovery of Glycolysis: the Embden-Meyerhof Glycolytic Pathway

8. Hans and Eduard Buchner (1860-1917) lay the cornerstone of modern biochemistry when they demonstrated that a cell-free yeast extract could convert glucose into ethanol and carbon dioxide just like viable yeast cells. DGo’ = -25.4 kcal . mol-1 C6H12O6 2 C2H5OH + 2 CO2

10. Glycolysis Controlled release of free energy DG, drives ADP ATP Does not require O2 Provides glycerol for lipid biosynthesis

11. steps1-5

12. 1. Hexokinase catalyzes: Glucose + ATPglucose-6-P + ADP ATP binds to the enzyme as a complex with Mg++. Mg++ interacts with negatively charged phosphate oxygen atoms, providing charge compensation & promoting a favorable conformation of ATP at the active site.

13. Coupling of reactions Example of “driven” reaction: Glc Glc 6-P Glc + P Glc 6-P (+3.3 kcal mol-1) ATP ADP + P (-7.3 kcal mol-1) Glc + ATP Glc 6-P + ADP ( DGo’ = -4 kcal . mol-1) (DG = - 8 kcal . mol-1)

14. Induced fit: Binding of glucose to Hexokinase induces a large conformational change. This brings the C6 OH of glucose close to the terminal Pi of ATP, & excludes water from the active site. It prevents catalysis of ATP hydrolysis rather than Pi transfer. It is a common motif for an enzyme active site to be at an interface between protein domains connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, following a substrate-induced conformational change.

15. Glycolysis Glucose 6-P (Glc 6-P): Glucose trapped after crossing membrane (because of its negative charge) Hexokinase is inhibited by its product Glc 6-P Glc 6-P mutated to Glc 1-P (polyssacharide biosynthesis) Glc 6-P oxydised to 6-P gluconate to drive NADP to NADPH Glc 6-P can be hydrolysed to Glc and exported from, e.g. a liver cell

16. steps1-5

17. Glycolysis Glc 6-P does not have the correct aldol structure for an aldol split. Therefore Glc 6-P is isomerised into fructose 6-phosphate (phosphoglucose isomerase: step 2). Before splitting another phosphoryl group from ATP is transferred to fructose 6-P giving rise to fructose 1,6-bisphosphate (addition of P destabilises Glc 6-P) The second phosphate means that each of the C3- products is phosphorylated to give GAP and DHAP (phosphofructokinase: step 3). Step 2

18. 2. Phosphoglucose isomerasecatalyzes: glucose-6-P (aldose) fructose-6-P (ketose) The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure.

20. The aldol condensation

21. Glycolysis Step 3 Second phosphorylation, introduces more ring strain assisting opening at step 4 (recharge repulsion) A more symmetrical structure that produces interchangeable products after ring cleavage at step 4

22. 3. Phosphofructokinase catalyzes: fructose-6-P + ATP fructose-1,6-bis-P + ADP This highly spontaneous reaction has a mechanism similar to that of Hexokinase. Phosphofructokinase, the rate-limiting step of Glycolysis, is highly regulated. Regulation will be discussed later.

23. 4. Aldolase catalyzes: fructose-1,6-bisphosphate  dihydroxyacetone-P + glyceraldehyde-3-P Note that carbons are renumbered in products of aldolase.

24. kJoules and not kcal!!!! = Phosphoglucose isomerase Conversion of Glc 6-P into two C3-compounds = +5.7 kcal mol-1 (GAP)

25. DG = Gº’ + 2.3 R T log10 [products] [reactants] All reactants are to be assumed to be present in the cell at 10-4 M DG = Gº’ + 2.3 R T log10 [GAP] [DHAP] [Fructose 1,6-P] DG = + 5.7 kcal mol-1 + 2.3 R T log10 [10-4][10-4] [10-4] DG = + 5.7 – 5.42 = +0.28 kcal. mol-1 The actual DG value at these assumed conc is compatible with free reversibility

26. 5. Triose Phosphate Isomerase(TIM) catalyzes: dihydroxyacetone-P glyceraldehyde-3-P Glycolysis continues from glyceraldehyde-3-P. TIM's K’eq favours dihydroxyacetone-P. Removal of glyceraldehyde-3-P by a subsequent spontaneous reaction allows throughput.

27. The ketose/aldose conversion involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase. Active site Glu and His residues extract and donate protons during catalysis. TIM is judged a "perfect enzyme." (see Stryer 5e, p 432). Reaction rate is limited only by the rate at which substrate & enzyme collide.

28. Triosephosphate Isomerase structure is an ab barrel, or TIM barrel. There are 8 parallel b-strands surrounded by 8 a-helices. Short loops connect alternating b-strands & a-helices. In a diverse array of enzymes, TIM barrels serve as scaffolds for active site residues, which are always located at the same end of the barrel, on the C-terminal ends of b-strands & loops connecting these to a-helices. There is debate whether the many different enzymes with TIM barrel structures are evolutionarily related.