Chapter 9: Glycolysis

GLYCOLYSIS A paradigm of metabolic regulation Under conditions of high ATP or citrate levels in cells, the catalytic activity of PFK-1 is normally inhibited owing to a high energy charge in the cells . 9.1 Overview of metabolism Catabolic pathways are the collection of enzymatic reactions in the cell that lead to the degradation of macromolecules and nutrients for the purpose of energy capture, usually in the form of ATP and reducing power In contrast, anabolic pathways use energy available from the hydrolysis of ATP and the oxidation of reducing equivalents to synthesize biomolecules for the cell The flux of metabolites through catabolic and anabolic pathways is determined by two primary factors: Essentially… 1. Level of enzyme activity, which is controlled by enzyme levels (gene transcription, Catabolic pathways protein synthesis, and protein turnover), break down large molecules into small catalytic activity (binding of regulatory molecules to release molecules and covalent modification), cellular energy, anabolic location (membranes or organelles) pathways use energy to 2. Level of substrates, which are obtained turn small molecules from the diet and released from energy into large molecules stores in the body Metabolite concentrations directly affect metabolic flux The overall change in free energy of the chemical reaction A → B in living cells is equal to the sum of the biochemical standard free energy change and the product of a value representing the gas constant, temperature, and actual concentrations of substrates and products If the concentration of product at equilibrium is much greater than the concentration of substrate, [B] >> [A], then the reaction A → B is highly favorable under standard conditions leading to the accumulation of B A large negative G indicates that under standard conditions, the reaction proceeds from left to right (A is converted to B), whereas a large positive G indicates that the reaction proceeds from right to left (B is converted to A) Often, metabolic reactions with G << 0 are used as a driving force to make reactions within a linked metabolic pathway proceed through use of shared intermediates Example: Glucose + Pi —> glucose-6-phosphate + H2O. ATP + H2O —> ADP + Pi. Glucose + ATP —> Glucose-6-phosphate + ADP. G = +13.8 kJ/mol G = -30.5 kJ/mol G = -16.7 kJ/mol Through reaction coupling, an unfavorable reaction may be driven forward by a favorable reaction Two types of data support the concept of metabolite channeling One is the finding that some glycolytic enzymes associate with each other in solution through high-affinity noncovalent interactions, suggesting that such complexes could form inside the cell The other evidence comes from kinetic data, which indicate that rates of coupled reactions in protein aggregates in vitro closely resemble in vivo reaction rates Such metabolic protein scaffolds would serve to increase the local concentration of metabolites and, moreover, could regulate enzyme activity through conformational changes induced by scaffold binding 9.2 Structures of Simple Sugars Glycolysis is basically the splitting of one molecule of glucose into two molecules of pyruvate. Thus, glucose is the first reactant in the glycolytic pathway Monosaccharides Aldehyde-containing monosaccharides . are called aldoses, whereas ketoneH O containing monosaccharides are called CH_OH c ketoses A carbon chiral center is an atom H C OH C O bonded to four different chemical groups Carbon Chiral compounds lack a plane of backbone symmetry and exist as two optical isomers, also called enantiomers Most monosaccharides in living organisms are of the D conformation; CHzOH CHOH L isomers do exist in nature, but they are usually conjugated to other Aldose Ketose molecules such as proteins or lipids Two monosaccharides that differ in the position of the hydroxyl group around only one carbon atom are called epimers If this bond is the result of a reaction between an alcohol group and the aldehyde group of an aldose sugar, it forms a hemiacetal; a bond between an alcohol group and the ketone group of a ketose sugar forms a hemiketal Disaccharides Disaccharides can contain different monosaccharide units connected through alpha or beta glycosidic bonds involving ring carbons The structures of three common disaccharides found in nature: .Lactose: Also called milk sugar, contains a beta (1 —> 4) glycosidic bond linking A galactose (Gal) to a glucose CHOH 0 Galactose to CHOH Sucrose: Made in plants and used as table sugar in its crystalline form, contains fructose (Fru) linked to glucose through the two anomeric carbons CHOH OH Ho Glucose OH CHOH Fructose CHOH OH OH Trehalose: A glucose disaccharide made in insects, contains a glycosidic bond between the two anomeric carbons to form the disaccharide GlucoseOH OH 0H yo yo GlucoseOH OH Hoke on Glucose 9.3 Glycolysis Generates ATP under Anaerobic Conditions Glycolysis, or the glycolytic pathway, is one of the core metabolic pathways in nature for three primary reasons: 1. Glycolytic enzymes are highly conserved among all living organisms, suggesting it is an ancient pathway 2. Glycolysis is the primary pathway for ATP generation under anaerobic conditions and in cells lacking mitochondria, such as erythrocytes 3. Metabolites of glycolysis are precursors for a large number of interdependent pathways, including mitochondrial ATP synthesis The glycolytic pathway consists of 10 enzymatic reactions The conversion of one molecule of glucose to two molecules of pyruvate by the glycolytic pathway occurs in the cytosol and generates two net molecules of ATP Pyruvate is transported into the mitochondrial matrix, where it is oxidized to CO2 and H2O under aerobic conditions, contributing to the generation of an additional 30 molecules of ATP The advantage is that a small amount of chemical energy in the form of 2 net ATP can be derived from glucose metabolism under conditions when O2 is limiting However, a yield of 2 net ATP from glycolysis alone under anaerobic conditions, compared to a total of 32 net ATP that can be generated from the complete oxidation of glucose under aerobic conditions, provides a yield of only 6% Because of the requirement for ATP hydrolysis in the initial reactions, followed by ATP synthesis in the later reactions, glycolysis is divided into two stages: Stage 1: ATP investment (reactions 1-5) Stage 2: ATP earnings reactions 6-10) 1. The enzyme . hexokinase, which Glucose is present in all 1 Hexokinase human cells, ATP phosphorylates glucose using ATP to form Glucose-6-Phosphate glucose-6phosphate 2 Phosphoglucoisomerase 2. Glucose-6phosphate is Fructose-6-Phosphate isomerized by phosphoglucoisoATP 3 Phosphofructokinase-1 merase in reaction 2 to Fructose-1,6-Biphosphate form fructose-6phosphate 4 Aldolase 3. In turn, fructose-6phosphate is a substrate for the phosphoryl Dihydroxyacetone-phosphate transfer reaction catalyzed by 5 Triose phosphate isomerase phosphofructokinase-1 in reaction 3 to Glyceraldehyde-3-phosphate (2) generate fructose-1,6biphosphate. The enzyme aldolase catalyzes an aldol cleavage reaction that splits the six-carbon fructose-1,6-BP intermediate 1,3-Biphosphoglycerate (2) into two phosphorylated products: glyceraldehyde-37 Phosphoglycerate kinase phosphate and ATP (2) dihydroxyacetone phosphate, each with three carbons 5. In the last reaction of stage 1, 3-Phosphoglycerate (2) dihydroxyacetone-P is isomerized by the enzyme triose phosphate isomerase, a 8 Phosphoglycerate mutase TIM barrel protein, to form a second molecule of glyceraldehyde-3-P 2-Phosphoglycerate (2) 6. Stage 2 of glycolysis begins with an oxidation reaction 9 Enolase catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase Phosphoenolpyruvate (2) 7. Reaction 7 is catalyzed by the enzyme phosphoglycerate kinase, which converts 1,310 Pyruvate kinase biphosphoglycerate to 3ATP (2) phosphoglycerate and represents the first of two Pyruvate (2) ATP generating steps in glycolysis 8. In reaction 8 of glycolysis, phosphoglycerate mutase isomerizes 3-phosphoglycerate to form 2-phosphoglycerate 9. The substrate listed above is Used for a dehydration reaction catalyzed by enolase in reaction 9 to generate phosphoenolpyruvate 10. In the final reaction of glycolysis, a second substrate-level phosphorylation takes place when the enzyme pyruvate kinase converts phosphoenolpyruvate to pyruvate . 6 Glyceraldehyde-3-phosphate dehydrogenase 4. In the table below that the reactions catalyzed by the enzymes hexokinase, phosphofructokinase-1, and pyruvate kinase have large negative delta G values and can therefore be considered irreversible reactions under physiologic conditions Reaction. Enzyme. Delta G Reaction. Enzyme. Delta G 1. Hexokinase -16.7 6. Glyceraldehyde-3-phosphate dehydrogenase +6.3 2. Phosphoglucoisomerase. +1.7 7. Phosphoglycerate kinase. -18.8 3. Phosphofructokinase-1. -14.2 8. Phosphoglycerate mutase. +4.6 4. Aldolase. +23.8 9. Enolase. +1.7 5. Triose phosphate isomerase. +7.5 10. Pyruvate kinase. -31.4 Stage 1 of the glycolytic pathway: ATP investment First, using ATP as the phosphoryl group donor, stage l reactions create phosphorylated compounds that are negatively charged and cannot diffuse out of the cell Reaction I: Phosphorylation of glucose by hexokinase or glucokinase The first reaction in glycolysis serves to activate glucose for catabolism by attaching a phosphoryl group to the hydroxyl group on C-6, generating glucose-6-P As with all kinase reactions, the ATP substrate for the reaction is in a complex with the divalent cation Mg2+, which functions to shield negative charges in the ATP molecule HO CH 0 ATP Ma OH ADP O CH ma o OH Hexokinase (or glucokinase) OH OH 0H OH 0 Hexokinase activity is inhibited by the product of the reaction, glucose-6-P, which accumulates in cells when flux through the glycolytic pathway is restricted These enzymatic properties of gluc kinase, along with its selective expression in specific cell types, facilitate its function as a metabolic "sensor" of high blood glucose levels Hexokinase binds glucose through an induced-fit mechanism that excludes H20 from the enzyme active site and brings the phosphoryl group of ATP into close proximity with C-6 of glucose Reaction 2: Isomerization of Glucose-6-P to Fructose-6-P by phosphoglucoisomerase Because the reaction has a small free energy change (G= -2.9 kJ/mol), the reaction is readily reversible and is controlled by the levels of glucose-6-P and fructose-6-P in the cell 0 O CH o O app OH 0 Phosphoglucoisomerase OH P O Cha CHOH o HO OH 0H 0 ma OH Reaction 3: Phosphorylation of fructose-6-P to fructose-1,6-BP by phosphofructokinase-1 Reaction 3 is the second ATP investment reaction in glycolysis. It couples ATP hydrolysis with a phosphoryl transfer reaction catalyzed by the enzyme phosphofructokinase-1 Phosphofructokinase-1 activity is allosterically controlled by the energy charge of the tell in such a way that increased AMP and ADP concentrations stimulate enzyme activity, whereas high ATP concentrations inhibit phosphofructokinase-1 activity 0 I O Cha ma ATP CH OH ADP 0 o HO OH o cha map CH 0 o o Phosphofructokinase-1 o HO OH OH All three effectors - AMP, ADP, and ATP - compete for binding to the same allosteric site on the enzyme, making AMP and ADP positive allosteric effectors and ATP a negative allosteric effector Reaction 4: Cleavage of fructose-1,6-BP into glyceraldehyde-3-P and dihydroxyacetone-P by aldolase 0 O CHz up CH 0 I 0 0 0 Aldolase Ho OH OH E yea Glyceraldehyde-3-P piggy P o b Dihydroxyacetone-P The mechanism of cleavage by aldolase involves the formation of a covalent enzyme-substrate Complex through the generation of a Schiff base requiring a lysine residue in the enzyme active site The four-step reaction can be summarized as follows: 1. The open-chain form of fructose-1,6-BP binds to the aldolase active site, and a lysine residue in the active site initiates a nucleophilic attack on the ketose carbon, generating a covalently bond protonated Schiff base intermediate 2. Base abstraction by an active site carbonyl group leads to C-C (aldol) cleavage between C-3 and C-4 of fructose, and a covalent enamine intermediate is formed. The first product, glyceraldehyde-3-P, is released 3. Isomerization leads to the formation of a second protonated Schiff base intermediate 4. The Schiff base intermediate is hydrolyzed, releasing the second product, dihydroxyacetone-P Reaction 5: Isomerization of dihydroxyacetone-P to glyceraldehyde-3-P by triose phosphate isomerase At the expense of investing 2 ATP, this stage produces two molecules of glyceraldehyde-3-P for every molecule Hig .IE of glucose that is phosphorylated in reaction 1 Stage 2 of the glycolytic pathway: ATP earnings it O Glyceraldehyde-3-P 0 1 0 Dihydroxyacetone-P Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-P to form 1,3-biphosphoglycerate by glyceraldehyde-3-P dehydrogenase The energy released from the oxidation of glyceraldehyde-3-P drives a phosphoryl group transfer reaction, using inorganic phosphate to produce 1,3-biphosphoglycerate Because NAD+ is required for the oxidation step in this reaction, NAD+ must be continually replenished within the cytosol to maintain flux through glycolysis H go 0 0 0 0 1o d NAD+ NADH + H+ 1 Triose phosphate isomerase Hey C H 0 0 Glyceraldehyde-3-P dehydrogenase Y Q 8 OH OH H 0 Enediol intermediate 0 8o In the first step, glyceraldehyde-3-P binds to the enzyme active site and reacts with the sulfhydryl group of a cysteine residue in the enzyme active site, forming a thiohemiacetal intermediate Reaction 7: Substrate-level phosphorylation to generate ATP in the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate by phosphoglycerate kinase Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because it replaces the 2 ATP that were used in stage 1 to prime the glycolytic pathway The high phosphoryl transfer energy present in the substrate is used to phosphorylase ADP to form ATP by the mechanism of substrate-level phosphorylation, leading to the conversion of 1,3-bisphosphoglycerate to 3phosphoglycerate o 0 it o ADP ATP Phosphoglycerate kinase 0 ftp.i 0 Substrate binding leads to a large conformational change in the enzyme that brings the substrates close together and excludes H2O from the active site The induced-fit mechanism of substrate binding maximizes accessibility to the enzyme active site without sacrificing the requirement that a hydrophobic environment be formed in the closed conformation Reaction 8: Phosphoryl shift in 3-phosphoglycerate to form 2-phosphoglycerate by phosphoglycerate mutase it 0 1 Iii ii Phosphoglycerate mutase JH o The reaction requires a phosphoryl transfer from a phosphorylated histidine residue (His-P) located in the enzyme active site Reaction 9: Dehydration of 2-phosphoglycerate to form phosphoenolpyruvate by enolase The arrangement of the phosphoryl group in phosphoenolpyruvate makes it much easier to transfer the phosphoryl group to ADP in the pyruvate kinase reaction However, when enolase converts 2-phosphoglycerate to phosphoenolpyruvate, it traps the phosphoryl group in an unstable enol form 050 0 H H2O o p O H GH 0ic o H2O o o C Enolase OH H H f 0 Reaction 10: Substrate-level phosphorylation to generate ATP in the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase In this reaction, the high phosphoryl transfer potential of phosphoenolpyruvate is used by the enzyme pyruvate kinase to generate pyruvate - the end product of glycolysis - and A net of 2 ATP for every glucose molecule entering the pathway oic 0 0 C 4 H 50 b ADP ATP Qc Pyruvate kinase 0 0 H Y H H 9.4 Regulation of the glycolytic pathway Rate-limiting reactions in a pathway are those reactions that proceed at the lowest rate compared to other reactions in the pathway Glycolysis and gluconeogenesis utilize the same enzymes for the six reversible steps in these two pathways, whereas irreversible steps are rate limiting and require three (glycolysis) or four (gluconeogenesis) pathway-specific enzymes to control metabolic flux through each pathway Glycolysis: catabolic pathway yielding 2 net ATP Gluconeogenesis: anabolic pathway consuming 4 ATP and 2 GTP Glucokinase is a molecular sensor of high glucose levels Four hexokinase genes have been identified in humans (hexokinase I-IV), all of which encode enzymes that are capable of converting glucose to glucose-6-P using the phosphoryl transfer energy of ATP In contrast, hexokinase IV, also known as glucokinase, has a low affinity for substrate, is highly specific for glucose, is expressed primarily in liver and pancreatic cells, and is not inhibited by glucose-6-P Moreover, because glucokinase is not inhibited by glucose-6-P, It is able to continue functioning even if flux through glycolysis cannot keep up with product formation This means that phosphorylation by glucokinase or hexokinase effectively retains glucose within the cell Another important function of glucokinase is to act as a glucose sensor and stimulate insulin release from pancreatic beta cells when blood glucose levels are elevated Allosteric control of phosphofructokinase-1 activity Phosphofructokinase-1 (PFK-1) is a well-characterized enzyme in the glycolytic pathway because of its vital role in controlling flux through the pathway In contrast, elevated levels of ATP, an indicator at high energy charge in the cell, and of citrate, which accumulates when the citrate cycle is inhibited, function as allosteric inhibitors of PFK-1 High ATP concentrations in the cell (high energy charge) increase the proportion of PFK-1 molecules in the inactive T-state conformation, which decreases the affinity of the enzyme active site for the substrate fructose-6-P In contrast, high AMP and ADP concentrations (low energy charge) increase the proportion of PFK-1 molecules in the active R-state conformation, which favors fructose-6-P binding Supply and demand of glycolytic intermediates Plants store polymeric glucose in starch granules, and animals that eat plants break down dietary starch into the disaccharide maltose using the salivary enzyme alpha-amylase 9.5 Metabolic fate of pyruvate Under aerobic conditions, the majority of pyruvate is metabolized in the mitochondria to CO2 and H2O by the citrate cycle and electron transport chain Under anaerobic conditions, such as occurs in muscle cells during strenuous exercise, pyruvate is converted to lactate by the enzyme lactate dehydrogenase A critical function of pyruvate metabolism in all three pathways is to regenerate the oxidized form f the coenzyme NAD+, which is required to maintain flux through the glyceraldehyde-3-P dehydrogenase reaction in glycolysis