Dr. DerVartanian is ill and will likely not be able to give lectures this week.

Transcription

1 Dr. DerVartanian is ill and will likely not be able to give lectures this week. Today s slides will be put on-line today, and are designed to introduce you to glycolysis. You should use these slides, along with Dr. DerVartanian s notes, and your reading of chapters (and chapter 6, pages ), TO STUDY THE MATERIAL and PREPARE for next week s exam.

2 Glycolysis and Citric Acid Cycle Glucose + 6 O 2 6 CO H 2 O Glucose = total ATPs during aerobic metabolism. + 6 Combustion: G Lost ~2800 kj mol -1 Glycolysis G stored 2NAD + +4e - 2NADH 2ADP 2ATP Citric Acid Cycle G stored 8NAD + +16e - 8NADH 2GDP(ADP) 2GTP(ATP) 2Q + 4e - 2QH

3 Glycolysis and Citric Acid Cycle hexose (6C) stage: 2 ATP s consumed. triose 2(3C) stage: 4 ATP s produced. Net: 2 ATP s. ALSO, 2 NADH Molecules and Pyruvate Net Reaction: Glucose + 2 ADP + 2 NAD Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H H 2 O Hexose Pyruvate catabolism produces most of the energy in mammalian cell! 8NAD + +16e - 8NADH ATP Equivalents ATP = 1 NADH = 2.5 (1.5)* QH 2 = 1.5 GTP = 1 Energy carriers (ATP/NADH) are in all life forms 2GDP(ADP) 2GTP(ATP) 2Q + 4e - 2QH 2 So, glucose = ATP s! Triose *It costs energy to transport NADH electrons into the mitochondria of some cells

4 Consume energy to setup Stage 2 Trap Glc in cell Essential Substrate for Stage 2

5 Glycolysis: Step 1, Hexokinase C6 Recall: hexokinase uses induced fit Glc Hexokinase drives passive transport of Glucose Passive Glc transporters in membrane Glc hexokinase Product Inhibition glucokinase Glc-6-P Metabolically irreversible rxn. It is inhibited by the G- 6P product AND Substrate Availability

6 Glycolysis: Step 2 Glucose 6-Phosphate Isomerase Aldose Ketose Ultimate cleavage site Stereospecific: uses -D-Glc-6P; produces -D-fructose-6P Aldose to ketose conversion Stereospecific: ring opening leads to ~30 % Beta G-6P, what drives the Beta to Alpha?

7 Step 3, Phosphofructokinase-1 PFK-1 First COMMITTED step of glycolysis Metabolically irreversible rxn under cellular conditions. It is an allosteric enzyme and a REGULATORY CONTROL step for glycolysis (ATP, AMP and citrate). (see page 299) ALSO Fructose-2,6-bisphosphate (F-2,6-BP)!!!!!!

8 Glycolysis: Step 4, Aldolase Rxn is near equilibrium K eq 1 10, 000 Reaction is very Unfavorable ( G = 5.7 kcal mol -1 ) Rapid depletion of 2 products in subsequent steps drives rxn

9 ONLY GAP can enter stage 2!. We must convert DHAP to a second molecule of GAP!

10 Step 5, Triose Phosphate Isomerase Ketose Aldose near equil. ONLY GAP can enter stage 2!

11 Stage 2: Energy Production ONLY GAP can enter stage 2! So far, we have consumed 2 molecules of ATP Energy Rich Molecules produced in stage 2: NADH ATP Pyruvate!!

12 Glycolysis: Step 6 Glyceraldehyde 3-Phosphate Dehydrogenase High Energy Mixed Anhydride Mixed anhydride of phosphoric acid and a carboxylic acid Redox RXNs: Follow the double bonds or the addition of an Oxygen. Generates NADH!!!!! Higher group transfer potential than ATP Redox reaction? Adds or removes a double bond OR Oxygen OR Sulfur atom Great Example of how enzymes use coupled reactions to do difficult chemistry: Formation of a high-energy molecule for making ATP!

13 NADH, the other energy carrier in the cell. NADH oxidation in the mitochondria produces 2.5 ATP molecules NAD + is a cellular oxidant! Carrying 2 e s

14 Glyceraldehyde 3-Phosphate Dehydrogenase: Coupled RXN 1) E Thioester High Energy 2) O Ester E Compare leaving groups! 1) oxidation 2) dehydration Coupled Rxn G G G Thioester covalent catalysis Traps the dg from oxidation to drive phosphorylation RXN Coord. RXN Coord. RXN Coord.

15 GAP Dehydrogenase (GAPDH) Nucleophilic Addition Thiohemiacetal Oxidation The thiohemiacetal oxyanion tautomerizes to produce an unstable carbanion, which is easily oxidized. 1,3-BPG NAD + 3-PG Nucleophilic Substitution NADH The favorable energy from the favorable oxidation is stored in the high energy thioester (yellow shading). The attacking phosphate substitutes the thiolate. What is the thiol pka?

16 High Energy Mixed Anhydride 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). Named for the reverse reaction

17 Glycolysis: Step 8,9 and 10 Acidic Proton Enolase will protonate the hydroxyl to form H 2 O High Energy phosphate Donor Phosphoenolpyruvate

18 If given you the enzyme names. You must know the reactions (be able to draw the sugar substrates and products) Know GAPDH: example of elegant coupled chemistry (GAPDH). But No ATP input!! Hexose Aldolase: largest uphill reaction (standard dg). What drives it? Triose phosphate isomerase. What drives it All energy producing and consuming steps Be able to draw the sugars if given a name. Know the names Triose What happens if there is no NAD +

19 Anaerobic Redox Balancing Getting rid of Electrons! Net Reaction: Glucose + 2 ADP + 2 NAD Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H H 2 O If a cell is not able to regenerate NAD + needed by GAPDH, glycolysis will stop! Lactate

20 Redox Balancing Getting rid of Electrons! Anaerobic Yeast Anaerobic Oxygen starved Muscles Aerobic Oxidative Phosphorylation NADH NAD + The Most efficient use of glucose! NEEDS a mitochondria!!

21 Gluconeogenesis: The Cori and Alanine (Cahill) Cycles (Liver). Also RBCs! The Cori Cycle converts Lactate produced in muscle during anaerobic respiration to glucose in the liver to control blood sugar. RBCs are also a major source of lactate (why?). During starvation, muscle protein catabolism produces energy and pyruvate. Pyruvate can be used to make OAA for Glucose and FA synthesis in the Liver via the Cahill Cycle. Cori cycle Alanine (Cahill) Cycle- pyruvate produced in muscle cannot be exported. In the Cahill Cycle, the amine groups of AA s are transferred to pyruvate to produce alanine. The alanine is exported from the muscle to the liver, where deamination produces pyruvate for gluconeogenesis.

22 Three Metabolically Irreversible Reactions 1. Hexokinase 3. Phosphofructokinase Pyruvate Kinase Will the pathway work based on the standard dg? Most reactions are near equilibrium in the cell, and have G close to zero * * * The table legend is wrong! Small +dg is NO PROBLEM as long as a larger dg is downstream!

23 Allosteric Control of Glycolysis and Gluconeogenesis Hexokinase Glucose-6-phosphatase We will learn about the Insulin and Glucagon response at the end of gluconeogenesis

24 Regulation of Hexokinase Passive Low affinity Glut 1 transporters in membrane Why is Hexokinase NOT a major control point in glycolysis? Glc Glc Insulin regulated High affinity Glut4 transporters. Glut4 inserts in membrane in response to insulin to increase Glc transport See Table 16.3 What drives Glc import? Product Inhibition hexokinase (glucokinase) Glc-6-P F6P PGI PFK-1 Glycolysis Regulation 1) Feedback Inhibition by product 2) Substrate Availability F-1,6-BP

25 Allosteric Control of Glycolysis and Gluconeogenesis Hexokinase Glucose-6-phosphatase Phosphofructokinase is the most important control site in mammalian glycolytic pathway. Enzyme is 340-kDa tetramer

26 Allosteric Regulation of PFK-1 Active Site Allosteric Site Activators *AMP- says ATP low, make more *F-2,6-BP * - says blood Glc High Inhibitors *ATP**- ATP stores are high *Citrate- CAC is stopped! *H + - Too much lactic acid! * *F-2,6-BP is NOT F-1,6-BP! WE WILL LEARN ABOUT THE IMPORTANT ROLE OF F-2,6-BP WHEN WE TALK ABOUT GLUCONEOGENESIS! Does it make sense that ATP is an allosteric inhibitor?

27 Fructose-2,6-biphosphate Allosteric Regulation of PFK-1 with AMP/ATP Look at the 0 F-2,6-BP High ATP saturation curve. [AMP] Why are Low concentrations of ATP required for PFK-1 to function? Why do High concentrations of ATP inhibit? Notice that the activator F-2,6-Bp can negate the inhibitory effect of high ATP. ATP is also substrate in the reaction! Active Site Allosteric Site

28 Allosteric Control of Glycolysis and Gluconeogenesis Hexokinase Glucose-6-phosphatase

29 Allosteric and Covalent Regulation of Pyruvate Kinase* WE WILL DISCUSS THE HORMONAL REGULATION OF PK at the END OF Gluconeogenesis! INHIBIT: When blood [Glc] drops, glucagon signals protein kinase A (PKA) to phosphorylate pyruvate kinase (Liver only) Allosteric Activators F-1,6-BP: says PFK-1 working Allosteric Inhibitors ATP- ATP stores are high Alanine- From the Cori Cycle/ gluconeogenesis is running ACTIVATE: When Blood sugar is high, Insulin signals phosphoprotein phosphatase 1 (PP1) to dephosphorylate pyruvate kinase. PP1 PKA *Named for the reverse reaction

30 Coordinated Regulation of glycolysis in muscle Contingent on glycogen levels! PFK-1: Key regulatory enzyme in glycolysis. We will learn more in gluconeogenesis!