CHAPTER 16 Glycolysis
Net reaction of Glycolysis Converts: 1 Glucose Hexose stage 2 pyruvate - Two molecules of ATP are produced - Two molecules of NAD + are reduced to NADH Triose stage Glucose + 2 ADP + 2 NAD + + 2 P i 2 Pyruvate + 2 ATP + 2 NADH + 2 H + + 2 H 2 O
Glycolysis can be divided into two stages Hexose stage 2 ATP are consumed per glucose Triose stage 4 ATP are produced per glucose NET: 2 ATP produced per glucose
Enzymes are needed to speed up the reactions. Hexose stage Note the near equilibrium rxns concentration affects flux Vs. Those that are irreversible points of regulation Triose stage
Enzymes are needed to speed up The reactions. Hexose stage Note the near equilibrium rxns concentration affects flux Vs. Those that are irreversible points of regulation Triose stage Two molecule of NADH are produced in triose stage and are equivalent to several ATP in terms of redox potential.
Enzyme 1 Hexokinase - Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to generate glucose 6-phosphate - Mechanism: Mg 2+ is an important cofactor, attack of C-6 hydroxyl oxygen of glucose on the g-phosphorous of MgATP 2- displacing MgADP - This is an irreversible reaction
Figure 16.2: Enzyme 1 Hexokinase 2 lobes show induced fit model
Enzyme 2 Phosphoglucose Isomerase - Converts glucose 6-phosphate (an aldose) to fructose 6-phosphate (a ketose) - Enzyme converts glucose 6-phosphate to open chain form of fructose 6-phosphate in the active site. A near equilibrium reaction
Enzyme 3 Phosphofructokinase-1 (PFK-1) - Catalyzes transfer of a phosphoryl group from ATP to the C-1 hydroxyl group of fructose 6-phosphate to form fructose 1,6-bisphosphate - PFK-1 is metabolically irreversible and a critical regulatory point for glycolysis in most cells (PFK-1 is the first committed step of glycolysis.
Enzyme 4 Aldolase - Aldolase cleaves the hexose, fructose 1,6-bisphosphate into two triose phosphates: glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) This reaction is also near equilibrium but products are favored
Enzyme 5 Triose Phosphate Isomerase - Conversion of dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP) This reaction is also near equilibrium but products are favored due to GAP metabolism
Fate of carbon atoms from hexose stage to triose stage Consumed 2 ATP in the process - per one glucose Figure 11.7
Enzyme 6 Glyceraldehyde 3-phosphate Dehydrogenase - Conversion of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphosphoglycerate (1,3-BPG) - One molecule of NAD + is reduced to NADH Oxidation and phosphorylation, yielding a high energy mixed acid hydride (NADH) NADH can be reoxidized elsewhere, such as the membrane associated electron transport chain
Enzyme 6 Glyceraldehyde 3-phosphate Dehydrogenase The coupling of these two reactions make the overall conversion near equilibrium Very unfavorable reaction
Enzyme 7 Phosphoglycerate Kinase - Uses the high-energy compound 1,3-bisphosphoglycerate to transfer a phosphoryl group to ADP to form ATP - 3-phosphoglycerate is formed in the process 1,3-BPG has more stored chemical energy than ATP
Enzyme 8 Phosphoglycerate Mutase - Catalyzes transfer of a phosphoryl group from one part of of the substrate molecule to another - Reaction occurs without input of ATP
Enzyme 8 Phosphoglycerate Mutase A different phosphate is placed on carbon-2.
Enzyme 9 Enolase: 2-phosphoglycerate to phosphoenolpyruvate (PEP) - Elimination of water (dehydration) yields PEP - PEP has a very high phosphoryl group transfer potential because it exists in its unstable enol form
Enzyme 10 Pyruvate Kinase: - Metabolically irreversible reaction - This reaction is another site of regulation - Pyruvate kinase is regulated both allosterically and by covalent modification with a phosphate group unstable
Triose Stage 2 glyceraldehyde 3- phosphate molecules generated per one glucose 2 NADH generated per one glucose 4 ATP generated per one glucose
Table 16.1: Irreversible reactions have large negative free energy (DG) When DG = 0, equilibrium is established
- For centuries, bakeries and breweries have exploited the conversion of glucose to ethanol and CO 2 by glycolysis in yeast Fates of pyruvate
Metabolism of Pyruvate 1. Aerobic conditions: pyruvate is oxidized to acetyl CoA which enters the citric acid cycle for further oxidation to CO 2 2. Anaerobic conditions: (microorganisms): pyruvate is converted to ethanol 3. Anaerobic conditions: (muscles, red blood cells): pyruvate is converted to lactate 4. Amino acid synthesis: pyruvate is a precursor to alanine
Anaerobic Conversion: Pyruvate to Ethanol - Yeast cells convert pyruvate to ethanol and CO 2 and oxidize NADH to NAD +. - Two steps (and enzymes) are required: - Decarboxylation to form acetaldehyde by pyruvate decarboxylase. - Coenzyme thiamine pyrophosphate(tpp) is also needed - Reduction of acetaldehyde to ethanol by alcohol dehydrogenase. This is coupled with the oxidation of NADH Redox is balance
Reduction of Pyruvate to Lactate muscles - anaerobic - Muscles lack pyruvate dehydrogenase and cannot produce ethanol from pyruvate - Muscle have lactate dehydrogenase to convert pyruvate to lactate
Reduction of Pyruvate to Lactate muscles - anaerobic This reaction regenerates NAD + for use by glyceraldehyde 3-phosphate dehydrogenase in glycolysis. This will maintain glycolytic flux. - Lactate formed in skeletal muscles during exercise is transported to the liver via the bloodstream. - Liver lactate dehydrogenase can convert lactate to pyruvate - Lactic acidosis can result from insufficient oxygen (an increase in lactic acid and decrease in blood ph)
Figure 16.7 Entry of other sugars into glycolysis From ingested fructose From ingested fructose
Figure 16.8:Fructose is converted to Glyceraldehyde 3-phosphate Conversion requires 2 ATP molecules same as the hexose stage for glucose.
Figure 16.9: Gal-1-P is activated by attachment of a UMP. Activated form Glu-6P phosphoglucomutase
Regulation of Glycolysis - Enzymes that are not reversible: Reaction 1 hexokinase Reaction 3 phosphofructokinase Reaction 10 pyruvate kinase - These steps are metabolically irreversible, and these enzymes are regulated. - All other steps of glycolysis are near equilibrium in cells and not regulated 1. When ATP is needed, glycolysis is activated - Inhibition of phosphofructokinase-1 is relieved by accumulation of AMP or fructose 2,6-bisphosphate - Pyruvate kinase is activated by fructose 1,6-bisphosphate 2. When ATP levels are sufficient, glycolysis activity decreases - Phosphofructokinase-1 is inhibited by ATP and Citrate - Hexokinase is inhibited by glucose 6-phosphate
Glucose transport into the cell- the first step in regulation Transporters are proteins consisting of ~500 AA single chain polypeptide (Integral membrane proteins) Insulin binding activates GLUT 4 embedded in plasma membrane. Glucose uptake into skeletal and heart muscles
Regulation of Hexokinase - Hexokinase reaction is metabolically irreversible - Glucose 6-phosphate (product) levels increase when glycolysis is inhibited at sites further along in the pathway - Glucose 6-phophate inhibits hexokinase
Regulation of Phosphofructokinase-1 (PFK-1) - ATP is a substrate and an allosteric inhibitor of PFK-1 - High concentrations of AMP (and ADP) allosterically activate PFK-1 by relieving the ATP inhibition. ([ATP] does not vary much in cells. [ADP] and [AMP]<< [ATP]) - Elevated levels of citrate in the liver (indicate ample substrates for citric acid cycle) also inhibit phosphofructokinase-1 Figure 16.12
Figure 16.13: Allosteric regulation of hexokinase and PFK
Glycolysis regulation in the Liver PFK-1 by Fructose 2,6-bisphosphate (F-2,6-BP) Fructose 2,6-bisphosphate is formed form fructose 6-phosphate and allosterically activates PFK-1 to increase its affinity for fructose 6-phosphate. PFK-2 PFK-1 Figure 16.14
Regulation of Liver Pyruvate Kinase allosteric effectors and covalent modification Protein Kinase A The hormone glucagon stimulates a protein kinase A, which phosphorylates pyruvate kinase, converting it to a less active form
Assignment Read Chapter 15 Read Chapter 16