Glycolysis. BCH 340 lecture 3 Chapter 8 in Lippincott 5 th edition

Transcription

1 Glycolysis B 40 lecture hapter 8 in Lippincott 5 th edition

2 All carbohydrates to be catabolized must enter the glycolytic pathway Glycolysis is degradation of glucose to generate energy (ATP) and to provide pyruvate (in the presence of oxygen) or lactate (in the absence of oxygen) Glycolysis is central in generating both energy and metabolic intermediates.

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4 Glycolysis takes place in the cytoplasm of all cells in the body but it is of physiological importance in: Tissues with no mitochondria: mature RBs, cornea and lens Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin and gastrointestinal tract Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise 4

5 Biological importance of glycolysis:. Energy production: Under anaerobic conditions: glycolysis gives ATP Under aerobic: glycolysis gives 8 ATP. xygenation of tissues: Through formation of, bisphosphoglycerate, which decreases the affinity of emoglobin to : Pure hemoglobin releases only 8% of oxygen to the tissues, however hemoglobin with,-bpg allows it to release 66% of the oxygen to the tissues. It is for this reason that hemoglobin, and not myoglobin, is more used in transferring oxygen between tissues and the lungs. 5

6 . Provides important intermediates: Dihydroxyacetone phosphate: can give glycerol- phosphate, which is used for synthesis of TGs and PLs (lipogenesis). Phosphoglycerate: which can be used for synthesis of amino acid serine. Pyruvate: which can be used in synthesis of amino acid alanine. 4. Aerobic glycolysis provides the mitochondria with pyruvate, which gives acetyl oa Krebs' cycle. 6

7 Steps: There are 0 enzyme-catalyzed reactions in glycolysis There are two stages Stage : (Reactions -5) A preparatory stage in which glucose is phosphorylated, converted to fructose which is again phosphorylated and cleaved into two molecules of glyceraldehyde- -phosphate. In this phase there is an investment of two molecules of ATP Stage : (reactions 6-0) The two molecules of glyceraldehyde-- phosphate are converted to pyruvate with concomitant generation of four ATP molecules and two molecules of NAD. Thus there is a net gain of two ATP molecules per molecule of Glucose in glycolysis. 7

8 4 6 5 glucose 6 P ATP ADP 5 4 Mg + exokinase glucose-6-phosphate. Phosphorylation of glucose: exokinase catalyzes: Glucose + ATP glucose-6-p + ADP ATP binds to the enzyme as a complex with Mg ++ A phosphoanhydride bond of ATP (~P) is cleaved ADP 8

9 4 6 5 glucose 6 P ATP ADP 5 4 Mg + The reaction catalyzed by exokinase is irreversible (glucose-6-p can not diffuse out of the cell because there are no specific carriers for phosphorylated sugars) This reaction is catalyzed by several isoenzymes of hexokinase and glucokinase: both requires Mg + as a cofactor exokinase glucose-6-phosphate 9

10 omparison between glucokinase and hexokinase enzymes: Site Affinity to glucose Substrate Effect of insulin Effect of glucose-6-p Function Liver only Glucokinase Low affinity (high km) i.e. it acts only in the presence of high blood glucose concentration. Glucose only Induces synthesis of glucokinase. No effect Acts in liver after meals. It removes glucose coming in portal circulation, converting it into glucose -6-phosphate. All tissue cells exokinase igh affinity (low km) i.e. it acts even in the presence of low blood glucose concentration. Glucose, galactose and fructose No effect Allosterically inhibits hexokinase It phosphorylates glucose inside the body cells. This makes glucose concentration more in blood than inside the cells. This leads to continuous supply of glucose for the tissues even in the presence of low blood glucose concentration.

11 4 6 5 P. Isomerization of glucose-6-p: 5 P Phosphoglucose Isomerase catalyzes: glucose-6-p (aldose) fructose-6-p (ketose) It is not rate-limiting or regulated step 6 4 Phosphoglucose Isomerase glucose-6-phosphate fructose-6-phosphate

12 Phosphofructokinase 6 P 5 6 P ATP ADP 5 P 4 fructose-6-phosphate Mg + 4 fructose-,6-bisphosphate. Phosphorylation of fructose-6-p Phosphofructokinase (PFK-) catalyzes: fructose-6-p + ATP fructose-,6-bisp + ADP Rate-limiting step PFK- is an allosteric enzyme, it is inhibited allosterically by elevated levels of ATP

13 P P fructose-,6- bisphosphate Aldolase P 4. leavage of fructose-,6-bisp: Aldolase catalyzes: fructose-,6-bisphosphate dihydroxyacetone-p + glyceraldehyde--p The reaction is reversible Aldolase A occurs in most tissues + P Aldolase B occurs in liver and kidney dihydroxyacetone glyceraldehyde-- phosphate phosphate Triosephosphate Isomerase

14 P P fructose-,6- bisphosphate Aldolase P 5. Isomerization of dihydroxyacetone phosphate: Triose Phosphate Isomerase (TIM) interconverts: dihydroxyacetone-p glyceraldehyde--p + Two molecules of glyceraldehyde--p produced for each glucose P dihydroxyacetone glyceraldehyde-- phosphate phosphate Triosephosphate Isomerase

15 Summary of First Stage of Glycolysis (Energy Investment) Glucose + ATP > GAP + ADP + + Recall that there are GAP per glucose

16 P glyceraldehyde- -phosphate Glyceraldehyde--phosphate Dehydrogenase + P i + + NAD + NAD P P,-bisphosphoglycerate 6. xidation of glyceraldehyde--phosphate Glyceraldehyde--phosphate Dehydrogenase catalyzes: glyceraldehyde--p + NAD + + P i,-bisphosphoglycerate + NAD + + igh energy compound

17 P glyceraldehyde- -phosphate Glyceraldehyde--phosphate Dehydrogenase + P i + + NAD + NAD This is the only step in Glycolysis in which NAD + is reduced to NAD NAD + is the cofactor in this reaction which acts as an oxidizing agent Glyceraldehyde--P Dehydrogenase is a tetrameric enzyme (one S in its active site) Glyceraldehyde--P Dehydrogenase is inhibited by iodoacetate P P,-bisphosphoglycerate

18 Phosphoglycerate Kinase P ADP ATP Mg + P,-bisphosphoglycerate P -phosphoglycerate 7. Formation of ATP from, BPG and ADP Phosphoglycerate Kinase catalyzes the Transfer of phosphoryl group from, bisphosphoglycerate to ADP generating ATP:,-bisphosphoglycerate + ADP -phosphoglycerate + ATP This phosphate transfer is reversible, since one ~P bond is cleaved & another synthesized

19 Phosphoglycerate Kinase P ADP ATP Mg + P P,-bisphospho- -phosphoglycerate glycerate molecules of ATP are produced (by Substrate-level phosphorylation) Recall every molecule of glucose gives rise to trioses!!!

20 Substrate level phosphorylation This means phosphorylation of ADP to ATP at the reaction itself In glycolysis there are examples: o. BPG + ADP Phosphoglycerate + ATP o PEP + ADP pyruvate + ATP

21 Phosphoglycerate Mutase P -phosphoglycerate P -phosphoglycerate 8. Shift of the P group from to Phosphoglycerate Mutase catalyzes the onversion of -phosphoglycerate to -phosphoglycerate (-PG). -phosphoglycerate -phosphoglycerate It is a freely reversible reaction

22 Enolase P 9. Dehydration of -P-glycerate to phosphoenolpyruvate Enolase catalyzes: -phosphoglycerate phosphoenolpyruvate + This dehydration reaction is Mg ++ -dependent and reversible Enolase is inhibited by fluoride P To measure glucose level in blood, fluoride is added to inhibit Enolase and stop glycolysis P -phosphoglycerate enolate intermediate phosphoenolpyruvate igh energy compound

23 P Pyruvate Kinase ADP ATP phosphoenolpyruvate pyruvate 0. Formation of pyruvate Pyruvate Kinase catalyzes the transfer of phosphoryl group from PEP to ADP generating ATP and Pyruvate phosphoenolpyruvate + ADP pyruvate + ATP This enzyme requires Mg ++ and K + Irreversible reaction

24 P Pyruvate Kinase ADP ATP phosphoenolpyruvate pyruvate This phosphate transfer from PEP to ADP is spontaneous (the free energy of PEP hydrolysis is coupled to the synthesis of ATP) This is the second substrate level phosphorylation reaction of glycolysis

25 Summary of Second Stage of Glycolysis GAP + NAD ADP + P i Pyruvate + NAD ATP

26 Summary of Glycolysis Glucose + NAD + + ADP + P i Pyruvate + NAD ATP can directly be used for doing work or synthesis NTE: NAD + must be regenerated for glycolysis to proceed!

27 Glycolysis Balance sheet for ~P bonds of ATP: ow many ATP ~P bonds expended? ow many ~P bonds of ATP produced? (Remember there are two fragments from glucose.) 4 Net production of ~P bonds of ATP per glucose:

28 Under the aerobic condition: Pyruvate is catabolized further in mitochondria through pyruvate dehydrogenase and citric acid cycle where all the carbon atoms are oxidized to. The free energy released is used in the synthesis of ATP, NAD and FAD.

29 Under anaerobic condition: In absence of oxygen, NAD+ + is not oxidized by the respiratory chain. Pyruvate is converted to Lactate in homolactic fermentation or in ethanol in alcoholic fermentation to regenerate NAD+. This helps continuity of glycolysis, as the generated NAD + will be used once more for oxidation of another glucose molecule (step6).

30 omolactic Fermentation: Lactate Dehydrogenase NAD + + NAD + pyruvate lactate Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense. Lactate dehydrogenase (LD) reduces pyruvate to lactate using NAD and thereby oxidizing it to NAD+ NAD + is regenerated by lactic fermentation to carry out GAPD reaction of glycolysis (step 6) ell membranes contain carrier proteins that facilitate transport of lactate

31 Lactate Dehydrogenase NAD + + NAD + pyruvate lactate Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs ycle or (in liver) converted back to glucose via gluconeogenesis

32 Lactate Dehydrogenase NAD + + NAD + pyruvate lactate Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment glucose to lactate and release it.

33 Alcoholic fermentation Pyruvate Decarboxylase Alcohol Dehydrogenase NAD + + NAD + pyruvate acetaldehyde ethanol Microorganisms and yeast convert pyruvate to ethanol, which is excreted as a waste product, and carbon dioxide to regenerate NAD + for glycolysis NAD is converted to NAD + in the reaction catalyzed by Alcohol Dehydrogenase.

34 Pyruvate Decarboxylase Alcohol Dehydrogenase NAD + + NAD + pyruvate acetaldehyde ethanol It is a two step process:. Pyruvate decarboxylase (PD) reaction: This enzyme is Mg ++ - dependent and requires an enzyme-bound cofactor, thiamine pyrophosphate (TPP). In this reaction a molecule of is released producing acetaldehyde.. Alcohol dehydrogenase reaction: Acetaldehyde is reduced to ethanol using NAD as reducing power, thus regenerating NAD +

35 Special features of glycolysis in RBs Mature RBs contain no mitochondria, thus: o They depend only upon glycolysis for energy production (= ATP). o Lactate is always the end product. Glucose uptake by RBs is independent on insulin hormone. Reduction of met-hemoglobin: Glycolysis produces NAD+ +, which used for reduction of met-hemoglobin in red cells. In most cells, bisphosphoglycerate is present in trace amount, but in erythrocytes it is present in significant amount:

36 In red cells, BPG is converted to,bpg which unites with oxy b and helps release of oxygen at tissues.