Pharm246 Glycolysis & Gluconeogenesis PDF

PHRM246 INTRO TO BIOCHEM CARBOHYDRATE METABOLISM PHRM246/PHARM SCIENCES/SHS/CHS/UKZN/Dr S Naidoo  GLYCOLYSIS & THE GLYCOLYTIC PATHWAY GLUCONEOGENESIS RELATIONSHIP BETWEEN GLYCOLYSIS & GLUCONEOGENESIS  CARBOHYDRATE METABOLISM Fates of die...

PHRM246 INTRO TO BIOCHEM CARBOHYDRATE METABOLISM PHRM246/PHARM SCIENCES/SHS/CHS/UKZN/Dr S Naidoo  GLYCOLYSIS & THE GLYCOLYTIC PATHWAY GLUCONEOGENESIS RELATIONSHIP BETWEEN GLYCOLYSIS & GLUCONEOGENESIS  CARBOHYDRATE METABOLISM Fates of dietary glucose The major source of dietary carbohydrate for humans is starch from consumed plant material. This is supplemented with a small amount of glycogen from animal tissue, disaccharides such as sucrose from products containing refined sugar and lactose in milk. Digestion in the gut converts all carbohydrate to monosaccharides which are transported to the liver and converted to glucose. The liver has a central role in the storage and distribution within the body of all fuels, including glucose. Glucose in the body undergoes one of three metabolic fates : It is catabolised to produce ATP This occurs in all peripheral tissues, particularly in brain, muscle and kidney. It is stored as glycogen This storage occurs in liver and muscle. It is converted to fatty acids Once converted to fatty acids, these are stored in adipose tissue as triglycerides. Glucose catabolism Glucose will be oxidised by all tissues to synthesise ATP. The first pathway which begins the complete oxidation of glucose is called glycolysis. GLYCOLYSIS This pathway cleaves the six carbon glucose molecule (C6H12O6) into two molecules of the three carbon compound pyruvate (C3H3O3-). This oxidation is coupled to the net production of two molecules of ATP/glucose. The diagram (next page) shows an outline of glycolysis. The full set of reactions and structures can be found in any biochemistry textbook.  One oxidation reaction occurs in the latter part of the pathway. It uses NAD as the electron acceptor. This co-factor is present only in limited amounts and once reduced to NADH, as in this reaction, it must be re- oxidised to NAD to permit continuation of the pathway. GLYCOLYSIS/AEROBIC CONDITIONS Anaerobic glycolysis pyruvate is reduced to a compound called lactate This single reaction occurs in the absence of oxygen (anaerobically) and is ideally suited to utilisation in heavily exercising muscle where oxygen supply is often insufficient to meet the demands of aerobic metabolism. The reduction of pyruvate to lactate is coupled to the oxidation of NADH to NAD. Aerobic metabolism of glucose (beyond pyruvate production) pyruvate is transported inside mitochondria and oxidised to a compound called acetyl coenzyme A (abbreviated to "acetyl CoA"). This is an oxidation reaction and uses NAD as an electron acceptor. By a further series of reactions (Citric acid cycle/TCA) acetyl CoA is oxidised ultimately to CO2. These reactions are coupled to a process known as the electron transport chain which has the role of harnessing chemical bond energy from a series of oxidation/reduction reactions to the synthesis of ATP and simultaneously re-oxidising NADH to NAD. Fast twitch muscle fibres utilise the first of the two mechanisms described above almost exclusively. Very heavily exercising muscle can use this pathway as the sole source of ATP synthesis for a short period of time. This probably evolved in humans as a defense mechanism, but is now used by athletes in sprint events. The formation of lactate as an end product from glucose extracts only a relatively small amount of the bond energy contained in glucose. Accumulation of lactate (actually lactic acid) also causes a reduction in intracellular pH. The lactate formed is removed to other tissues and dealt with by one of two mechanisms: a) it is converted back to pyruvate. The pyruvate then proceeds to be further oxidised by the second mechanism described above, finally producing a large amount of ATP. b) it is converted back to glucose in the liver Biochemistry of Metabolism Glycolysis  6 CH OPO 2- 2 3 5 O H H H 4 H 1 OH OH OH 3 2 H OH glucose-6-phosphate Glycolysis takes place in the cytosol of cells. Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially there is energy input corresponding to cleavage of two ~P bonds of ATP.  6 CH2OH 6 CH OPO 2- 2 3 ATP ADP 5 O 5 O H H H H H H 4 1 4 H 1 OH H OH Mg2+ OH OH OH OH 3 2 3 2 H OH Hexokinase H OH glucose glucose-6-phosphate 1. Hexokinase catalyzes: Glucose + ATP à glucose-6-P + ADP The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP. ATP binds to the enzyme as a complex with Mg++.  NH2 ATP N N adenosine triphosphate N O O O N - O P O P O P O CH2 adenine O - - - H H O O O H H OH OH ribose Mg++ interacts with negatively charged phosphate oxygen atoms, providing charge compensation & promoting a favorable conformation of ATP at the active site of the Hexokinase enzyme.  6 CH2OH 6 CH OPO 2- Induced fit mechanisms: 5 O ATP ADP 5 2 3 O H H H H Glucose binding 4 H OH H 1 2+ 4 H OH H 1 Mg to Hexokinase stabilizes a OH 3 2 OH OH 3 2 OH conformation in which: H OH Hexokinase H OH glucose glucose-6-phosphate the C6 hydroxyl of the bound glucose is close to the terminal phosphate of glucose ATP, promoting catalysis. Hexokinase water is excluded from the active site. This prevents the enzyme from catalyzing ATP hydrolysis, rather than transfer of phosphate to glucose.  6 CH2OH 6 CH OPO 2- 2 3 ATP ADP 5 O 5 O H H H H H H 4 1 4 H 1 OH H 2+ OH Mg OH OH OH OH 3 2 3 2 H OH Hexokinase H OH glucose glucose-6-phosphate The reaction catalyzed by Hexokinase is highly spontaneous. A phospho-anhydride bond of ATP (~P) is cleaved. The phosphate ester formed in glucose-6-phosphate has a lower DG of hydrolysis. 2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose) ßà fructose-6-P (ketose) Acid/base catalysis, with ring opening isomerization via an enediolate intermediate, and then ring closure. 3. Phosphofructokinase catalyzes: fructose-6-P + ATP à fructose-1,6-bisP + ADP This highly spontaneous reaction has a mechanism similar to that of Hexokinase. The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated  2- 1CH2OPO 3 2C O H O 2- HO 3C H Aldolase 3 CH 2 OPO 3 1C H 4C OH 2C O + H 2C OH 2- H C OH 1CH2OH 3 CH2OPO 3 5 2- 6 CH 2 OPO 3 dihydroxyacetone glyceraldehyde-3- phosphate phosphate fructose-1,6- bisphosphate Triosephosphate Isomerase 4. Aldolase catalyzes: fructose-1,6-bisphosphateßà dihydroxyacetone-P + glyceraldehyde-3-P The reaction is an aldol cleavage, the reverse of an aldol condensation. Note that C atoms are renumbered in products of Aldolase.  2- 1 CH2OPO3 2C O H O 2- HO 3 C H Aldolase 3 CH2OPO 3 1C H 4C OH 2C O + H 2C OH 2- H C OH 1CH2OH 3 CH2OPO3 5 2- dihydroxyacetone glyceraldehyde-3- 6 CH2OPO 3 phosphate phosphate fructose-1,6- bisphosphate Triosephosphate Isomerase 5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P ßà glyceraldehyde-3-P Glycolysis continues from glyceraldehyde-3-P. TIM's Keq favors dihydroxyacetone-P. Removal of G-3-P by a subsequent spontaneous reaction allows throughput.  Triosephosphate Isomerase H H OH H O + + + + H C OH H H C H H C C O C OH H C OH CH2OPO32- CH2OPO32- CH2OPO32- dihydroxyacetone enediol glyceraldehyde- phosphate intermediate 3-phosphate The ketose/aldose conversion involves acid/base catalysis, via an enediol intermediate Active site Glu and His residues on enzyme extract and donate protons during catalysis.  Glyceraldehyde-3-phosphate Dehydrogenase H O + H+ O OPO32- 1C NAD+ NADH 1C + Pi H C OH H C OH 2 2 2- 2- 3 CH2 OPO3 3 CH2 OPO3 glyceraldehyde- 1,3-bisphospho- 3-phosphate glycerate 6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi ßà 1,3-bisphosphoglycerate + NADH + H+  Glyceraldehyde-3-phosphate dehydrogenase H O + H+ O OPO32- + 1C NAD NADH 1C + Pi H C OH H C OH 2 2 2- 2- 3 CH2OPO3 3CH2OPO3 glyceraldehyde- 1,3-bisphospho- 3-phosphate glycerate Oxidation of the aldehyde in glyceraldehyde-3-phosphate, to a carboxylic acid, drives formation of an acyl phosphate, a "high energy" bond (~P). This is the only step in Glycolysis in which NAD+ is reduced to NADH.  Phosphoglycerate Kinase O OPO32- ADP ATP O O- 1C 1 C H 2C OH H 2C OH 2- Mg2+ 2- 3 CH 2 OPO 3 3 CH 2 OPO 3 1,3-bisphospho- 3-phosphoglycerate glycerate 7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP ßà 3-phosphoglycerate + ATP This phosphate transfer is reversible (low DG), since one ~P bond is cleaved & another synthesized. The enzyme undergoes substrate-induced conformational change similar to that of Hexokinase.  Phosphoglycerate Mutase O O- O O- 1 C 1 C H 2C OH H 2C OPO32- 2- 3 CH 2 OPO 3 3 CH2OH 3-phosphoglycerate 2-phosphoglycerate 8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate ßà 2-phosphoglycerate Phosphate is shifted from the OH on C3 to the OH on C2  Enolase O - H+ - O - OH- O- O O O C C 1 C 1 H 2 C OPO32- C OPO32- 2C OPO32- 3 CH2OH CH2OH 3 CH2 2-phosphoglycerate enolate intermediate phosphoenolpyruvate 9. Enolase catalyzes: 2-phosphoglycerate ßà phosphoenolpyruvate + H2O This dehydration reaction is Mg++-dependent. 2 Mg++ ions interact with oxygen atoms of the substrate carboxyl group at the active site. The Mg++ ions help to stabilize the enolate anion intermediate that forms when a Lys extracts H+ from C2.  Pyruvate Kinase O O- O O- ADP ATP 1 C 1 C 2 C OPO32- 2 C O 3 CH2 3 CH3 phosphoenolpyruvate pyruvate 10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP à pyruvate + ATP  Pyruvate Kinase O O- O O- O O- C ADP ATP C C 1 1 1 2 C OPO32- C 2 OH 2 C O 3 CH2 3 CH2 3 CH3 phosphoenolpyruvate enolpyruvate pyruvate This phosphate transfer from PEP to ADP is spontaneous. w Removal of Pi from PEP yields an unstable enol, which spontaneously converts to the keto form of pyruvate. K+ and Mg++ required at the active site of Pyruvate Kinase.  glucose Glycolysis ATP Hexokinase ADP glucose-6-phosphate Phosphoglucose Isomerase fructose-6-phosphate ATP Phosphofructokinase ADP fructose-1,6-bisphosphate Aldolase glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate Triosephosphate Isomerase glycolysis continued …  glyceraldehyde-3-phosphate NAD+ + Pi Glyceraldehyde-3-phosphate NADH + H+ Dehydrogenase glycolysis continued… 1,3-bisphosphoglycerate ADP Phosphoglycerate Kinase ATP 3-phosphoglycerate Phosphoglycerate Mutase 2-phosphoglycerate H2O Enolase phosphoenolpyruvate ADP Pyruvate Kinase ATP pyruvate  Glycolysis Balance sheet for ~P bonds of ATP: 2 w How many ATP ~P bonds expended? ________ w How many ~P bonds of ATP produced? (Remember 4 there are two 3C fragments from glucose.) ________ w Net production of ~P bonds of ATP per glucose: 2 ________ Balance sheet for ~P bonds of ATP: w 2 ATP used w 4 ATP produced (2 from each of two 3C fragments from glucose) w Net production of 2 ~P bonds of ATP per glucose. Glycolysis - total pathway, omitting H+: glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 pyruvate + 2 NADH + 2 ATP In aerobic organisms: w pyruvate produced in Glycolysis is oxidized to CO2 via Krebs Cycle w NADH produced in Glycolysis & Krebs Cycle is re-oxidized via the respiratory chain, with production of much additional ATP.  Glyceraldehyde-3-phosphate Dehydrogenase H O + H+ O OPO32- Fermentation: 1C NAD+ NADH 1C + Pi H C OH H C OH 2 2 Anaerobic organisms lack a 3 CH2OPO3 2- 3CH2OPO3 2- respiratory chain. glyceraldehyde- 1,3-bisphospho- 3-phosphate glycerate They must reoxidize NADH produced in Glycolysis through some other reaction, because NAD+ is needed for the Glyceraldehyde-3-phosphate Dehydrogenase reaction. Usually NADH is re-oxidized when pyruvate is converted to a more reduced compound. The complete pathway, including Glycolysis and the re-oxidation of NADH, is called fermentation.  Lactate Dehydrogenase O O- O O- C NADH + H+ NAD+ C C O HC OH CH3 CH3 pyruvate lactate E.g., Lactate Dehydrogenase catalyzes reduction of the keto in pyruvate to a hydroxyl, yielding lactate, as NADH is oxidized to NAD+. Lactate, in addition to being an end-product of fermentation, serves as a mobile form of nutrient energy, & possibly as a signal molecule in mammalian organisms. Cell membranes contain carrier proteins that facilitate transport of lactate.  Lactate Dehydrogenase O O- O O- C NADH + H+ NAD+ C C O HC OH CH3 CH3 pyruvate lactate Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense. Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted back to pyruvate (via lactate dehydrogenase), which may be oxidized in Krebs Cycle or (in liver) converted back to glucose via gluconeogenesis  Lactate Dehydrogenase O O- O O- C NADH + H+ NAD+ C C O HC OH CH3 CH3 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. Lactate taken up by adjacent neurons is converted to pyruvate that is oxidized via Krebs Cycle.  Pyruvate Alcohol Decarboxylase Dehydrogenase O O- CO2 H NADH + H+ NAD+ H C O C O C H C OH CH3 CH3 CH3 pyruvate acetaldehyde ethanol Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product. NADH is converted to NAD+ in the reaction catalyzed by Alcohol Dehydrogenase. Glycolysis, omitting H+: glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 pyruvate + 2 NADH + 2 ATP Fermentation, from glucose to lactate: glucose + 2 ADP + 2 Pi à 2 lactate + 2 ATP Anaerobic catabolism of glucose yields only 2 “high energy” bonds of ATP.  DGo' DG Glycolysis Enzyme/Reaction kJ/mol kJ/mol Hexokinase -20.9 -27.2 Phosphoglucose Isomerase +2.2 -1.4 Phosphofructokinase -17.2 -25.9 Aldolase +22.8 -5.9 Triosephosphate Isomerase +7.9 negative Glyceraldehyde-3-P Dehydrogenase -16.7 -1.1 & Phosphoglycerate Kinase Phosphoglycerate Mutase +4.7 -0.6 Enolase -3.2 -2.4 Pyruvate Kinase -23.0 -13.9 *Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John Wiley & Sons, New York, p. 613. REGULATION OF GLYCOLYSIS Flux through the Glycolysis pathway is regulated by control of 3 KEY enzymes that catalyze spontaneous reactions: Hexokinase, Phosphofructokinase & Pyruvate Kinase. w Local control/regulation of metabolism involves varying concentrations of pathway substrates or intermediates, to benefit the cell. w Global control is for the benefit of the whole organism, & often involves hormone-activated signal cascades. Liver cells have major roles in metabolism, including maintaining blood levels various of nutrients such as glucose. Thus global control especially involves liver. Some aspects of global control by hormone-activated signal cascades will be discussed later.  6 CH2OH 6 CH OPO 2- 2 3 ATP ADP 5 O 5 O H H H H H H 4 1 4 H 1 OH H 2+ OH Mg OH OH OH OH 3 2 3 2 H OH Hexokinase H OH glucose glucose-6-P Hexokinase is inhibited by product glucose-6-phosphate: w by competition at the active site w by allosteric interaction at a separate enzyme site. In the liver, when glucose product is too high, cells trap glucose by phosphorylating it, preventing exit on glucose carriers. Product inhibition of Hexokinase ensures cells don’t continue to accumulate glucose from the blood, if [glucose-6- phosphate] within the cell is sufficient.  6 CH2OH 6 CH OPO 2- Glucokinase is 2 3 ATP ADP 5 O 5 O H H H H a variant of 4 H OH H 1 4 H OH H 1 Hexokinase 2+ Mg OH OH OH OH 3 2 3 2 found in liver. H OH Hexokinase H OH glucose glucose-6-phosphate Glucokinase has a high KM for glucose. § It is active only at high [glucose]. One effect of insulin, a hormone produced when blood glucose is high, is activation in liver of the gene that codes for Glucokinase enzyme. Unlike hexokinase, glucokinase is not subject to product inhibition by glucose-6-phosphate. Liver will take up & phosphorylate glucose even when liver [glucose-6-phosphate] is high.  Glucokinase is subject to inhibition by glucokinase regulatory protein (GKRP). The ratio of Glucokinase: GKRP in liver changes in different metabolic states, providing a mechanism for modulating glucose phosphorylation. Glucokinase, Glycogen Glucose with high KM for glucose, Hexokinase or Glucokinase Glucose-6-Pase allows liver to store glucose Glucose-1-P Glucose-6-P Glucose + Pi as glycogen in the fed state Glycolysis Pathway when blood [glucose] is Pyruvate high. Glucose metabolism in liver Glucose-6-phosphatase catalyzes release of Pi from glucose-6- P. Thus glucose is released from the liver to the blood as needed to maintain blood [glucose]. The enzymes Glucokinase & Glucose-6-phosphatase, both found in liver but not in most other body cells, allow the liver to control blood [glucose].  Pyruvate Kinase O O- O O- Pyruvate Kinase, the last step in C ADP ATP C 1 1 Glycolysis, is controlled in liver C OPO32- C O 2 2 partly by modulation of the 3 CH2 3 CH3 amount of enzyme. phospho-enolpyruvate pyruvate High [glucose] within liver cells causes a transcription factor carbohydrate-responsive-element-binding protein (ChREBP) to be transferred into the nuclei, where it activates transcription of the gene for Pyruvate Kinase. This facilitates converting excess glucose to pyruvate, which is metabolized to acetyl-CoA, the main precursor for synthesis of fatty acids, for long term energy storage.  Phosphofructokinase 6 CH OPO 2- 1CH2OH 6 CH OPO 2- 1CH2OPO32- 2 3 2 3 O ATP ADP O 5 H HO 2 5 H HO 2 H 4 3 OH Mg2+ H 4 3 OH OH H OH H fructose-6-phosphate fructose-1,6-bisphosphate Phospho-fructo-kinase is usually the rate-limiting step of the Glycolysis pathway. Phosphofructokinase is allosterically inhibited by ATP. w At low ATP concentration, the substrate ATP binds only at the active site. w At high ATP concentration, ATP binds also at a low-affinity regulatory site, promoting the tense conformation.  Glycogen Glucose Hexokinase or Glucokinase Glucose-1-P Glucose-6-P Glucose + Pi Glycolysis Glucose-6-Pase Pathway Pyruvate Glucose metabolism in liver Inhibition of the Glycolysis enzyme Phospho-fructokinase when [ATP] is high prevents breakdown of glucose in a pathway whose main role is to make ATP. It is more useful to the cell to store glucose as glycogen when ATP is plentiful.  Gluconeogenesis The process of conversion of lactate to glucose is called gluconeogenesis, uses some of the reactions of glycolysis (but in the reverse direction) and some reactions unique to this pathway to re-synthesize glucose. This pathway requires an energy input (as ATP) but has the role of maintaining a circulating glucose concentration in the bloodstream (even in the absence of dietary supply) and also maintaining a glucose supply to fast twitch muscle fibres.