陳泰源 博士 中央研究院 生物化學研究所 2007/5/1 National Taiwan Normal University

1. 陳泰源 博士 中央研究院 生物化學研究所 2007/5/1 National Taiwan Normal University

3. Glycogen, Starch, Sucrose Storage Pentose phosphate pathway (oxidation) Glycolysis (oxidation) Pyruvate Ribose 5-phosphate

4. 14.1 Glycolysis http://www.tcd.ie/Biochemistry/IUBMB-Nicholson/swf/glycolysis.swf

5. Glycolysis • The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla(腎髓), brain, and sperm. • Fermentation is a general term for the anaerobicdegradation of glucose or other organic nutrients to obtain energy (ATP). • The pathway are conserved evolutionary in vertebrates, yeast and spinach.

9. 1. phosphorylation of glucose G 6-P

10. Kinase: (transferases): catalyze the transfer of the terminal phosphoryl group from ATP to some acceptor nucleophile. • Hepatocytes (Liver cell) also contain a form of hexokinase called hexokinase IV or glucokinase (more specific for glucose and differs from other hexokinase in kinetic and regulatory properties • Isozymes: Two enzymes that catalyze the same reaction but are encoded in different genes

11. 2. Isomerization of glucose 6-phosphate G 6-P F 6-P

12. The phosphohexose isomerase reaction • the ring opening and closing reactions (steps 1 and 4 ) are catalyzed by an active-site His residue. • The movement of the proton between C-2 and C-1 (steps 2 and 3 ) is base-catalyzed by an active-site Glu residue (shown as B:). The proton (pink) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and the nearby hydroxyl group. • After its transfer from C-2 to the active-site Glu residue, the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step 2 is not necessarily the same one that is added to C-1 • Step 3 (The additional exchange of protons (yellow and blue) between the hydroxyl groups and solvent is shown for completeness. The hydroxyl groups are weak acids and can exchange protons with the surrounding water whether the isomerization reaction is underway or not.)

13. 3. Phosphorylation of fructose 6-phosphate: the first committed step in glycolysis F 6-P F 1,6-BP

14. Phosphofructokinase-1 (PFK-1)-- fructose 6-phosphate to fructose 1, 6 bisphosphate. Irreversible under cellular condition. • PFK-2: fructose 6-phosphate to fructose 2, 6 bisphosphate. • Major regulatory enzyme in glycolysis – committed step - In some organisms, fructose 2,6-bisphosphate is a potent allosteric activator of PFK-1. • The enzyme (PFK-1) is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. When increases??

15. 4. Cleavage of fructose 1,6-bisphosphate G 3-P F 1,6-BP DHAP

16. 5. Interconversion of the triose phosphate

17. Interconversion of the Triose phosphates— End of preparatory phase of glycolysis

18. 6. Oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate 1,3-BPG

19. 7. Phosphoryl transfer from 1,3-bisphosphoglycerate to ADP 3-PGA

20. The formation of ATP by phosphoryl group transfer from a substrate is referred to as a substrate-level phosphorylation Substrate-level phosphorylation soluble enzymes chemical intermediates Respiration-linked phosphorylation Photophosphorylation membrane-bound enzymes transmembrane gradients of protons

21. Substrate-level phosphorylation

22. Respiration-linked phosphorylation or Photophosphorylation ATP ADP H+ H+

23. Glyceraldehyde 3-phosphate dehydrogenase and Phosphoglycerate kinase are coupled in vivo • Glyceraldehyde 3-phosphate dehydrogenase catalyzes an endergonic reaction while phosphoglycerate kinase catalyzes an exergonic reaction. • When these two reactions are coupled (which happens in vivo), the overall reaction is exergonic.

24. 8. Conversion of 3-phosphoglycerate to 2-phosphoglycerate 2-PGA

25. The phosphoglycerate mutase reaction

26. The phosphoglycerate mutase reaction COO- | HCOH | CH2O Phospho-glycerate mutase 2,3-bisphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate

27. 9. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate PEP

28. 10. Transfer of the phosphoryl group from phosphoenolpyruvate to ADP

30. Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi  2 pyruvate + 2ADP + 2NADH + 2H+ + 4ATP + 2H2O Glucose + 2ADP + 2NAD+ + 2Pi  2 pyruvate + 2ATP + 2NADH + 2H+ 在有氧狀況下，產生的NADH很快就被送到mitochondria中用來合成ATP

31. Questions?? • The fate of the carbon skeleton of glucose • The input of Pi and ADP and the output of ATP • The pathway of electrons in the oxidation-reduction reactions

32. 14.2 Feeder Pathways for Glycolysis

33. Feeder Pathways for Glycolysis 2 2 1 2 3 Phosphorolysis 2 Glycolysis 3 3 Glycolysis Glycolysis

34. phosphoglucomutase 1 Glycogen and Starch Are Degraded by Phosphorolysis • Phosphorolysis: Glycogen (animal tissues and in microorganisms) is degraded by glycogen phosphorylase (attack by Pi)at a nonreducing end to generate glucose 1P; repetitively until it approaches an (1, 6) branch point • starch (plants) use starch phosphorylase in the same way.

35. p.536

36. p.536 Branch point (a16) is removed by debranching enzyme

37. Transferase activity of Debranching enzyme -1,6 glucosidase activity of Debranching enzyme P P P P P P P P phosphorylase

38. Dietary Polysaccharides and Disaccharides Undergo Hydrolysis to Monosaccharides 2 • Starch is the major source of carbohydrates; firstly salivary a amylasebreaks glycosidic bond water, not Pi, is the attacking species. • Pancreas a-amylase continues the breakdown process - maltose and maltotriose (the di- and trisaccharides of (1→4) glucose) and limit dextrins, fragments of amylopectin containing (1→6) branch points. • Maltose and dextrins are degraded by enzymes of the intestinal brush border (the fingerlike microvilli of intestinal epithelial cells, which greatly increase the area of the intestinal surface).

39. Dietary polysaccharides and disaccharides are hydrolyzed to monosaccharides • Lactose intolerance — lactose cannot be completely digested and absorbed in the small intestine, and in the large intestine the lactose is converted by bacteria into toxic products that cause abdominal cramps and diarrhea. • Complication- undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring the retention of water in the intestine.

40. 3 Conversion of galactose to glucose 1-phosphate • from the intestine to the liver - Galactose is phosphorylated at C-1 by galactokinase firstly . • UDP galactose, which is formed when galactose 1P displaces glucose 1P from UDP-glucose by UDP-glucose; galactose 1P uridylyltransferase. • UDP-galactose is then converted by UDP-glucose 4-epimerase to UDP-glucose, in a reaction that involves oxidation of C-4 by NAD, then reduction of C-4 by NADH; the result is inversion of the configuration at C-4. • The UDP glucose is recycled through another round of the same reaction - conversion of galactose 1P to glucose 1P; there is no net production or consumption of UDP-galactose or UDP-glucose. Glycolysis

41. Epimer and epimerase (p. 241) • Two sugars that differ only in the configuration around one carbon atom are called epimers. • Enzymes that catalyze inversion of the configuration about an asymmetric carbon in a substrate having more than one center of asymmetry are called epimerases.

42. Galactosemia - 半乳糖血(症) • Galactokinase deficiency galactosemia, high galactose concentrations are found in blood and urine. Infants develop cataracts(白內障), caused by deposition of the galactose metabolite Galactitol in the lens - strict limitation of galactose in the diet. • Transferase-deficiency galactosemia - poor growth in children, speech abnormality, mental deficiency, and liver damage that may be fatal. • Epimerase-deficiency galactosemia leads to similar symptoms

43. 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation

44. Three possible catabolic fates of the pyruvate 2 1 3 CH16

45. 1. Pyruvate is the terminal electron acceptor in lactic acid fermentation • When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of pyruvate and NADH produced in glycolysis. • NAD+ is regenerated from NADH by the reduction of pyruvate to lactate (lactate dehydrogenase) • The lactate formed by active skeletal muscles can be recycles; --- in blood to liver and converted to glucose (Cori Cycle)

46. p.538 Fermentation • Fermentation is referring to the process when no oxygen is consumed or no change in the concentration of NAD+ or NADH during energy extraction. • Fermentation is a general term for the anaerobicdegradation of glucose or other organic nutrients to obtain energy (ATP).

47. The purpose of fermentation is to regenerate NAD+ 2 ADP 2 ADP 2 ADP • In order to continue regenerating NAD+, cells come up a strategy. • During fermentation, NAD+ is regenerated during the reduction of pyruvate, the product of glycolysis. glucose glucose glucose 2 pyruvate 2 pyruvate 2 pyruvate glycolysis glycolysis glycolysis 2 NADH 2 NADH 2 NADH 2 ATP 2 ATP 2 ATP 2 NAD+ 2 NAD+ 2 NAD+ 2 NADH 2 NADH 2 NADH 2 pyruvate 2 pyruvate 2 pyruvate fermentation fermentation fermentation 2 lactate 2 lactate 2 lactate 2 NAD+ 2 NAD+ 2 NAD+

48. 1 2 3 2. Ethanol is the reduced product in alcohol fermentation • Yeast and other microorganisms ferment glucose to ethanol and CO2, • The CO2 produced by pyruvate decarboxylation in brewer’s yeast is responsible for the characteristic carbonation of champagne. • In baking, CO2 released by pyruvate decarboxylase when yeast is mixed with a fermentable sugar causes dough to rise. • Pyruvate decarboxylase (not oxidation); is absent in vertebrate tissues. • Alcohol dehydrogenase (human liver) oxidation of ethanol from ingested or produced by intestinal microorganisms (in company with the reduction of NAD+ to NADH) 1 (human liver)

49. Thiamine pyrophosphate (TPP) is the coenzyme of pyruvate decarboxylase • Thiamine pyrophosphate is derived from vitamin B1 (thiamine). • Lack of vitamine B1 will lead to beriberi (edema, pain, paralysis, death; Singhalese “I cannot” Signifying the person is too ill to do anything.).

50. Fermentations Yield a Variety of Common Foods and Industrial Chemicals • Yogurt (Lactobacillus bulgaricus) – lactic acid; the resulting drop in pH; Swiss cheese (Propionibacterium freudenreichii), - propionic acid and CO2; pickles, sauerkraut, sausage, soy sauce, kimchi (Korea)… • drop in pH associated with fermentation also helps to preserve foods. • Clostridium acetobutyricum ferments starch to butanol and acetone. • Industrial fermentations: These fermentations are generally carried out in huge closed vats in which temperature and access to air are adjusted to favor the multiplication of the desired microorganism and to exclude contaminating methanol, formic, acetic, propionic, butyric, and succinic acids, and glycerol, ethanol, isopropanol, butanol, and butanediol. • Some industrial fermentations – immobilize the cells in an inert support, to pass the starting material continuously through the bed of immobilized cells, and to collect the desired product in the effluent.