Carbohydrate Catabolism Figure 24.5

Glycolysis

Glycolysis Start materials End products Arrow type No. of reactions Type of reactions Enzyme name for each reaction Condition of each reaction Intermediates name and No. of there carbon Functional group of each metabolites ATP production/ consumption

Glycolysis Start materials End products Arrow type No. of reactions Type of reactions Enzyme name for each reaction Condition of each reaction Intermediates name and No. of there carbon Functional group of each metabolites ATP production/ consumption

Glycolysis What is the overall pathway in glycolysis? How is the 6-carbon glucose converted to the 3-carbon glyceraldehyde-3-phosphate? How is glyceraldehyde-3-phosphate converted to pyruvate? How is pyruvate metabolized anaerobically? Where are the control points in the glycolytic pathway? How much energy can be produced by glycolysis?

Glycolysis Definition Location Reaction of glycolysis Glycolysis is the sequence of reactions that converts glucose into pyruvate in the presence of oxygen (aerobic) or lactate in the absence of oxygen (anaerobic) with the production of ATP Location Glycolysis is the major pathway for the utilization of glucose and is found in cytosol of all cells Reaction of glycolysis The breakdown of glucose to pyruvate is brought about by sequential action of 10 enzymes which can be divided into two phases First phase, energy requiring phase or preparative phase Second phase, energy generating phase pay off phase

Glycolysis A three-phase pathway in which: Glucose is oxidized into pyruvic acid (PA) It loses 2 pairs of hydrogens NAD+ is reduced to NADH + H+ It accepts 2 pairs of hydrogens lost by glucose ATP is synthesized by substrate-level phosphorylation Pyruvic acid: end-product of glycolysis Moves on to the Krebs cycle in an aerobic pathway (i.e. sufficient oxygen available to cell) Is reduced to lactic acid in an anaerobic environment (insufficient O2 available to cell) pyruvic acid lactic aicd

Glycolysis: Phase 1 and 2 Phase 1: Sugar activation Two ATP molecules activate glucose into fructose-1,6-diphosphate The 1 and 6 indicate which carbon atom to which they are attached. Phase 2: Sugar cleavage (splitting) Fructose-1,6-bisphosphate (6 C’s) is split into two 3-carbon compounds: Glyceraldehyde 3-phosphate (GAP)

Glycolysis: Phase 3 Phase 3: Oxidation and ATP formation The 3-carbon sugars are oxidized (reducing NAD+); i.e., 2 H’s + NAD NADH2 Inorganic phosphate groups (Pi) are attached to each oxidized fragment The terminal phosphates are cleaved and captured by ADP to form four ATP molecules The final products are: Two pyruvic acid molecules Two NADH + H+ molecules (reduced NAD+) A net gain of two ATP molecules

Stage 1 Glucose F-1,6-BP Stage 2 DHAP GAP Stage 3 2X PEP Pyruvate

Glycolysis

The overall pathway of glycolysis Glycolysis is the first stage of glucose metabolism One molecule of glucose is converted to fructose-1,6-bisphosphate, which gives rise to two molecules of pyruvate Glycolysis plays a key role in the way organisms extract energy from nutrients Once pyruvate is formed, it has one of several fates

The reactions of glycolysis What are the reaction of glycolysis? In glycolysis, glucose is converted to pyruvate in a multistep pathway. Glucose is converted to pyruvate in a series of 10 reactions, only one of which is an oxidation

The reactions of glycolysis (cont’d) Phosphorylation of glucose to give glucose-6-phosphate Isomerization of glucose-6-phosphate to give fructose-6-phosphate Phosphorylation of fructose-6-phosphate to yield fructose-1,6-bisphosphate Cleavage of fructose-1,6,-bisphosphate to give glyceraldehyde-3-phosphate and dihyroxyacetone phosphate Isomerization of dihyroxyacetone phosphate to give glyceraldehyde-3-phosphate

The reactions of glycolysis (cont’d) Oxidation of glyceraldehyde-3-phosphate to give 1,3-bisphosphoglycerate Transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP to give 3-phosphoglycerate Isomerization of 3-phosphoglycerate to give 2-phosphoglycerate Dehydration of 2-phosphoglycerate to give phosphoenolpyruvate Transfer of a phosphate group from phosphoenolpyruvate to ADP to give pyruvate

Conversion of glucose to glyceraldehyde-3-phosphate (preparation phase) of glycolysis In step 1 of glycolysis, glucose is phosphorylated to give glucose-6-phosphate G6P is an intermediate in several metabolic pathway The reaction is endergonic, as it is driven by the free energy of hydrolysis of ATP The kinase is ATP dependent enzymes that transfer a phosphate group from ATP to a substrate hexokinas Glucokinase This reaction is irreversible (control point)

Conversion of glucose to glyceraldehyde-3-phosphate (cont,d) The second step is the isomerization of glucose-6-phosphate to fructose-6-phosphate The C-1 aldehyde of glucose-6-phosphate is reduced to a hydroxyl group The C-2 hydroxyl group is oxidized to give the ketone group of fructose-6-phosphate There is no net redox reaction Isomerase is an enzyme that catalyzes the structural rearrangement of isomer

Conversion of glucose to glyceraldehyde-3-phosphate (cont,d) Step 3 Fructose-6-phosphate is then phosphorylated by ATP again to generate fructose-1,6-bisphosphate This is the second reaction to be coupled to ATP hydrolysis This is the key point reaction (F1,6BP key intermediate) This reaction is irreversible (control point)

Conversion of glucose to glyceraldehyde-3-phosphate (cont,d) In step 4 Fructose-1,6-bisphosphate is split into two 3-carbon fragments Reaction catalyzed by aldolase Side chains of an essential Lys and Cys play key roles in catalysis the reaction aldolase

Conversion of glucose to glyceraldehyde-3-phosphate (cont,d) In step 5, dihydroxyacetone phosphate (DHAP) is converted to glyceraldehyde-3-phosphate These compounds are trioses

Summary In the first stages of glycolysis, glucose is converted to two molecules of glyceraldehyde-3-phosphate The key intermediate in this series of reactions is fructose-1,6-bisphosphate. The reaction that produces this intermediate is a key control point of the pathway, and the enzyme that catalyzes it, phosphofructokinase, is subject to allosteric control

Glyceraldehyde-3-Phohsphate is Converted to Pyruvate (pay off phase) Step 6 The first reaction that begins the conversion to pyruvate involves the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate This reaction involves addition of a phosphate group, as well as an electron transfer reaction The oxidizing agent, NAD+, is reduced to NADH Dehydrogenase is an enzyme that oxidizes a substrate by transferring one or more hydrides (H-) to an acceptor, usually NAD+/NADP+ or FAD/FMN

Oxidation and Phosphorylation Reaction

Oxidation and Phosphorylation Reaction

Glyceraldehyde-3-Phohsphate is Converted to Pyruvate (Cont’d) 1,3-bisphosphoglycerate is converted to 3 phosphoglycerate This step (step 7) involves another reaction in which ATP is produced by phosphorylation of ADP 1,3-bisphosphoglycerate transfers a phosphate group to ADP. This is known as substrate-level phosphorylation Reaction is catalyzed by phosphoglycerate kinase This reaction is the sum of the endergonic phosphorylation of ADP and the exergonic hydrolysis of the mixed phosphate anhydride

Glyceraldehyde-3-Phohsphate is Converted to Pyruvate (Cont’d) The next step (step 8) involves the isomerization of 3-phosphoglycerate to 2-phosphoglycerate The phosphate group is transferred from carbon 3 to carbon 2 of the glyceric acid backbone This reaction is catalyzed by phosphoglyceromutase Mutase is an enzyme that catalyzes the intramolecular shift of a chemical group from one position to another within the same molecule such as phosphoryl group

Glyceraldehyde-3-Phohsphate is Converted to Pyruvate (Cont’d) Next, (step 9) 2-phosphoglycerate loses one molecule of water, producing phosphenolpyruvate Enolase catalyzes the dehydration reaction (-H2O) and requires a Mg2+ as a cofactor Phosphoenolpyruvate contains a high energy bond enolase

Glyceraldehyde-3-Phohsphate is Converted to Pyruvate (Cont’d) Phosphenolpyruvate (PEP) transfers its phosphate group to ADP, producing ATP and pyruvate Reaction is catalyzed by pyruvate kinase The double bond shift to the oxygen on carbon 2 and a hydrogen shifts to carbon 3 This reaction is irreversible (control point)

Summary In the final stages of glycolysis, two molecules of pyruvate are produced for each molecule of glucose that entered the pathway These reactions involve electron transfer (redox step 6), and the net production of two ATP for each glucose There are three control points in the glycolytic pathway

Glucose and other monosaccharides Enzyme location cytosol Entering substrates Glucose and other monosaccharides Enzyme location cytosol Net ATP production 2 ATP / glucose enter the pathway Coenzyme production 2NADH + 2H+ formed under aerobic conditions Final products under aerobic conditions Pyruvate under anaerobic conditions Lactate Net reaction Aerobic: Anaerobic:

Fates of pyruvate from glycolysis

Anaerobic Metabolism of Pyruvate Under anaerobic conditions, the most important pathway for the regeneration of NAD+ is reduction of pyruvate to lactate Lactate dehydrogenase (LDH) is a tetrameric isoenzyme consisting of H and M subunits; H4 predominates in heart muscle, and M4 in skeletal muscle

The conversion of pyruvate to lactate in muscles NAD+ Needs to be Recycled to Prevent Decrease in Oxidation Reactions So NAD + will be present for further glycolysis to take place

Alcoholic Fermentation Two reactions lead to the production of ethanol: Decarboxylation of pyruvate to acetaldehyde Reduction of acetaldehyde to ethanol Pyruvate decarboxylase is the enzyme that catalyzes the first reaction This enzyme require Mg2+ and the cofactor, thiamine pyrophosphate (TPP) Alcohol dehydrogenase catalyzes the conversion of acetaldehyde to ethanol

Alcoholic fermentation NAD+ Needs to be Recycled to Prevent Decrease in Oxidation Reactions So NAD + will be present for further glycolysis to take place

Summary Pyruvate is converted to lactate in anaerobic tissues, such as actively metabolizing muscle. NAD+ is recycled in the process In some organisms, pyruvate is converted to ethanol in a process requiring thiamine pyrophosphate as a coenzyme

Control Points in Glycolysis Three reactions exhibit particularly large decreases in free energy; the enzymes that catalyze these reactions are sites of allosteric control Hexokinase (step 1) Phosphofructokinase (step 3) Pyruvate kinase (step 10)

Energy yield from glycolysis The ATP yield from glycolysis is different in anaerobic and aerobic conditions During aerobic (oxygen plenty) condition, the two NADH generated in step 6 can enter the mitochondrial electron transport chain to complete oxidation As each NADH provides 3ATPs, this reaction provides 3 x 2 = 6 ATPs The net gain of energy from glycolysis pathways is 8 ATPs

Energy yield (number of ATP generated) per molecule of glucose in the glycolytic pathway, under aerobic conditions (oxygen is available) Step Reaction Enzyme Source No. of ATPs gained /glucose molecule 1 Hexokinase - - 1 3 Posphofructokinase 6 Glyceraldehyde-3-phosphate dehydrogenase NADH 3 X 2 = 6 7 1,3-bisPhosphoglycerate kinase ATP 1 x 2 = 2 10 Pyruvate kinase Total = 10 minus 2 = 8

Energy yield from glycolysis (cont’d) But when oxygen is in deficiency (anaerobic condition)one molecule of glucose is converted to two molecule of lactate, the net yield of 2 molecules of ATP 4 molecules of ATP are synthesised by the 2 substrate level phosphorylation (steps 7 and 10) But 2 ATP molecules are used in the steps 1 and 3, the net yield is only 2 ATP

Energy yield (number of ATP generated) per molecule of glucose in the glycolytic pathway, under anaerobic conditions (oxygen deficiency) Step Enzyme Source No. of ATPs gained /glucose molecule 1 Hexokinase - - 1 3 Posphofructokinase 7 1,3-bisPhosphoglycerate kinase ATP 1 x 2 = 2 10 Pyruvate kinase Total = 4 minus 2 = 2

Glucosephosphate isomerase 2 ATP 1 Hexokinase G-6-P Glucosephosphate isomerase 2 F-6-P ATP 3 Phosphofructokinase F-1,6-BP 4 Aldolase Triosphosphate isomerase DHAP GA3P 5 6 Glyceraldehyde-3-phosphate dehydrogenase 1,3BPG ATP NADH 7 3-Phosphoglycerate kinase 3-PG 8 Phosphoglyceromutase 2-PG 9 Enolase PEP ATP 10 Pyruvate kinase Pyruvate

What did you learn? Answer the following questions regarding glycolysis and energy. What is the overall energy yield in ATP production when one molecule of glucose is converted to pyruvate? Based on free energy yield, predict the reactions that are irreversible or poorly reversible in the glycolysis pathway. Why is the phosphoglycerate kinase reaction reversible? What about the other kinase reactions? Explain why G-6-P is ismerized to F-6-P, which then phosphorylated again to fructose F1,6-bP utilizing another ATP

Practice 4 Q 4 During glycolysis, a 6-carbon sugar diphosphate molecule is split into two 3-carbon sugar phosphate molecules. A) True B) False   Q 5 Under aerobic conditions, the end-product of glycolysis is further reduced to yield more ATP.

Answer to practice 4 Q 4 During glycolysis, a 6-carbon sugar diphosphate molecule is split into two 3-carbon sugar phosphate molecules. A) True B) False   Q 5 Under aerobic conditions, the end-product of glycolysis is further reduced to yield more ATP.

Practice 1 Q 1 In glycolysis, glucose is converted to A) CO2 and H2O. B) pyruvate. C) citrate. D) acetyl coA. E) NAD+ and ADP.

Practice 2 Q 2 The NET result of a single glycolysis run is the formation of A) 1 NADH and 1 ATP. B) 2 NADH and 2 ATP. C) 2 NADH and 4 ATP. D) 4 NADH and 2 ATP. E) 4 NADH and 4 ATP.

Answer to practice 2 Q 2 The NET result of a single glycolysis run is the formation of A) 1 NADH and 1 ATP. B) 2 NADH and 2 ATP. C) 2 NADH and 4 ATP. D) 4 NADH and 2 ATP. E) 4 NADH and 4 ATP.

Practice 3 Q 3 Under anaerobic conditions, the end-product of glycolysis is converted to A) CO2 and H2O. B) amino acids. C) lactic acid. D) hydrochloric acid. E) acetic acid.

Answer to practice 3 Q 3 Under anaerobic conditions, the end-product of glycolysis is converted to A) CO2 and H2O. B) amino acids. C) lactic acid. D) hydrochloric acid. E) acetic acid.