Glycolysis Overview - Organic Chemistry

Organic Chemistry II Md Golam Moula, Ph.D.  GLYCOLYSIS Breakdown of glucose to pyruvate Intracellular site: occurs in cytoplasm Types: Aerobic glycolysis: glucose to pyruvate to acetyl CoA Then TCA, oxidative phosphorylation Anaerobic glycolysis: glucose to...

Organic Chemistry II Md Golam Moula, Ph.D.  GLYCOLYSIS Breakdown of glucose to pyruvate Intracellular site: occurs in cytoplasm Types: Aerobic glycolysis: glucose to pyruvate to acetyl CoA Then TCA, oxidative phosphorylation Anaerobic glycolysis: glucose to pyruvate to lactate RBC: anaerobic glycolysis RBC do not have mitochondria 4 Glycolysis Overview 5 Energy investment and harvest phase E n e r g y 6 Glycolysis Overview 7  Reaction Steps 1. Hexokinase catalyzes: Glucose + ATP  glucose-6-P + ADP 2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose)  fructose-6-P (ketose) 3. Phosphofructokinase catalyzes: fructose-6-P + ATP  fructose-1,6-bisP + ADP 4. Aldolase catalyzes: fructose-1,6-bisphosphate  dihydroxyacetone-P + glyceraldehyde-3-P 5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P  glyceraldehyde-3-P 6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi  1,3-bisphosphoglycerate + NADH + H+ 7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP  3-phosphoglycerate + ATP 8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate  2-phosphoglycerate 9. Enolase catalyzes: 2-phosphoglycerate  phosphoenolpyruvate + H2O 10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP  pyruvate + ATP 8  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. 1. Hexokinase catalyzes: Glucose + ATP  glucose-6-P + ADP 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 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++. 2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose)  fructose-6-P (ketose) 6 CH OPO 2 2 3 5 6 CH OPO 2 1 CH2OH H O H 2 3 O H 4 H 1 5 H HO 2 OH OH OH H 4 3 OH 3 2 OH H H OH Phosphoglucose Isomerase glucose-6-phosphate fructose-6-phosphate The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure. A similar reaction catalyzed by Triosephosphate Isomerase will be presented in detail. 3. Phosphofructokinase catalyzes: fructose-6-P + ATP  fructose-1,6-bisP + ADP 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 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, as will be discussed later. 4. Aldolase catalyzes: fructose-1,6-bisphosphate  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.  lysine 1CH2OPO3 2 H  2C NH (CH2)4 Enzyme H3N+ C COO + HO 3 CH CH2 H 4 C OH CH2 H 5 C OH CH2 2 6 CH2OPO3 CH2 Schiff base intermediate of  NH3 Aldolase reaction A lysine residue at the active site functions in catalysis. The keto group of fructose-1,6-bisphosphate reacts with the amino group of the active site lysine, to form a protonated Schiff base intermediate. Cleavage of the bond between C3 & C4 follows.  5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P  glyceraldehyde-3-P 2 1CH2OPO3 2C O H O 2 HO 3C H Aldolase 3 CH2 OPO 3 1C H 4C OH 2C O + H 2C OH 2 H C OH 1CH2OH 3 CH2OPO3 5 2 6 CH2 OPO 3 dihydroxyacetone glyceraldehyde-3- phosphate phosphate fructose-1,6- bisphosphate Triosephosphate Isomerase Glycolysis continues from glyceraldehyde-3-P. TIM's Keq favors dihydroxyacetone-P. Removal of glyceraldehyde-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, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase. Active site Glu and His residues are thought to extract and donate protons during catalysis. 6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi  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 Exergonic 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.  7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP  3-phosphoglycerate + ATP Phosphoglycerate Kinase O OPO32 ADP ATP O O 1C 1 C H 2C OH H 2C OH 2 Mg2+ 2 3 CH2 OPO 3 3 CH2 OPO 3 1,3-bisphospho- 3-phosphoglycerate glycerate This phosphate transfer is reversible (low G), since one ~P bond is cleaved & another synthesized. The enzyme undergoes substrate-induced conformational change similar to that of Hexokinase. 8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate  2-phosphoglycerate Phosphoglycerate Mutase O O O O C 1 C 1 H 2C OH H 2C OPO32 2 3 CH2 OPO 3 3 CH2OH 3-phosphoglycerate 2-phosphoglycerate Phosphate is shifted from the OH on C3 to the OH on C2.  9. Enolase catalyzes: 2-phosphoglycerate  phosphoenolpyruvate + H2O 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 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 C #2. 10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP  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.  PEP has a larger G of phosphate hydrolysis than ATP.  Removal of Pi from PEP yields an unstable enol, which spontaneously converts to the keto form of pyruvate. Required inorganic cations K+ and Mg++ bind to anionic residues at the active site of Pyruvate Kinase.  Glucose Phosphorylation Energy requirements: If glucose were phosphorylated with inorganic phosphate, The energy required would be 3.3 kcal/mol. So the reaction is coupled with one that releases energy: ATP is hydrolysed, releasing energy: G0 = -7.3 kcal/mol. glucose + ATP-> glucose 6P + ADP The net energy is a release: G0 = -4 kcal/mol. This is how the chemical energy of ATP is used to do biochemical work. 22  Energy yield of Glycolysis Aerobic Glycolysis: Glucose to pyruvate: 2 ATP/mol of glucose + 2 NADH Anaerobic Glycolysis: Glucose to lactate: 2 ATP/mol of glucose (red blood cells, exercising muscles, white blood cells, kidney medulla, eye) 23 24  REGENERATION OF NAD+ Availability of NAD+ is necessary for glycolysis to continue – In anaerobic conditions (e.g., in RBCs and exercising muscles) Electrons are transferred from NADH to pyruvate by lactate dehydrogenase, forming NAD+ and lactate RBCs lack mitochondria and therefore depend entirely on anaerobic glycolysis for energy needs. In muscle tissue under hypoxic conditions, the energy needs of the tissue are partially supplied by anaerobic glycolysis. Lactate build-up during anaerobic glycolysis limits the extent to which muscle can obtain energy by this means. Accumulation of lactic acid causes a decrease in muscle cell pH Decreased pH interferes with function of the contractile machinery of the muscle. Elevated muscle lactate accounts for fatigue and pain induced by strenuous exercise. 25  TRANSPORT OF REDUCING EQUIVALENTS Inner mitochondrial membrane is impermeable to NADH No transport protein can translocate NADH across this membrane directly NADH formed in glycolysis enters mitochondria for oxidation via one of the two shuttles 1. Glycerol phosphate (glycerol 3-P) shuttle 2. Malate–aspartate shuttle 26 GLYCEROL 3-PHOSPHATE SHUTTLE Mitochondrial Glycerol 3-phosphate dehydrogenase (Similar to Complex II) Co Q Cytosolic Glycerol 3-phosphate dehydrogenase 27 GLYCEROL 3-PHOSPHATE SHUTTLE 28 GLYCEROL 3-PHOSPHATE SHUTTLE 29 GLYCEROL 3-PHOSPHATE SHUTTLE 30  NADH reduces dihydroxyacetone phosphate to glycerol 3-phosphate in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase The reverse reaction is catalyzed by an integral membrane flavoprotein (FAD) that transfers electrons to CoQ of the electron transport chain The oxidation of NADH using this pathway leads to the generation of 1.5 ATP instead of 2.5 ATP. The loss of energy makes this process irreversible. Therefore, the number of ATP produced differs depending on whether the NADH produced by glycolysis takes the glycerol phosphate shuttle or malate shuttle. 5 ATP for glycerol phosphate shuttle and 7 ATP for malate shuttle. 31  MALATE– ASPARTATE SHUTTLE The re-oxidation of malate in the mitochondrial matrix generates NADH that can pass electrons to the electron-transport chain and provide 2.5 ATP Completion of the shuttle cycle requires the activities of mitochondrial and cytosolic aspartate transaminase. The process is driven by accumulation of NADH in the cytosol and the utilization of NADH in the mitochondria. No energy is required. 32