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BCHEM 253 – METABOLISM IN HEALTH AND DISEASES Lecture 5 Bioenergetic Principles and the ATP cycle – Glycolysis Christopher Larbie, PhD Introduction D-Glucose is a major fuel for most organisms. D-Glucose metabolism occupies the center position for all metabolic pathways. Glucose contains a great deal of potential energy. The complete oxidation of glucose yields −2,840 kJ/mol of energy. Glucose + 6O 2 → 6CO2 + 6H2 O ΔGo’ = −2,840 kJ/mol Glucose also provides metabolic intermediates for biosynthetic reactions. Bacteria can use the skeletal carbon atoms obtained from glucose to synthesize every amino acid, nucleotide, cofactor and fatty acid required for life. For higher plants and animals there are three major metabolic fates for glucose. Nearly every living cell catabolizes glucose and other simple sugars by a process called glycolysis. Glycolysis differs from one species to another only in the details of regulation and the fate of pyruvate. Glycolysis is the metabolic pathway that catabolizes glucose into two molecules of pyruvate. 1|P a g e ©Dr. Christopher Larbie Glycolysis Glycolysis occurs in the cytosol of cells and is essentially an anaerobic process since the pathway’s principal steps do not require oxygen. The glycolytic pathway is often referred to as the Embden- Meyerhof pathway in honor of the two of the three biochemical pioneers (What about Otto Warburg?) who discovered it. The glycolytic pathway is shown below. Glycolysis consists of 10 enzyme catalyzed reactions. The pathway can be broken down into two phases. The first phase encompasses the first five reactions to the point that glucose is broken down into 2 molecules of glyceraldehyde 3-phosphate. Phase 1 consumes two molecules of ATP. The second phase includes the last five reactions converting glyceraldehyde 3-phosphate into pyruvate. Phase 2 produces 4 molecules of ATP and 2 molecules of NADH. The net reaction (Phase 1 + Phase 2) produces 2 molecules of ATP and 2 molecules of NADH per molecule of glucose. Phase 1 - Preparatory Phase 1. The first reaction of glycolysis is the transfer of a phosphoryl group from ATP to glucose. 2. The second reaction is the isomerization of glucose 6-phosphate to fructose 6phosphate. 3. The third step is the first committed step of glycolysis. This reaction is the transfer of a second phosphoryl group from ATP to fructose 6-phosphate. 4. The fourth step of glycolysis involves the aldolytic cleavage of the C3-C4 bond to yield two triose phosphates. 5. The 5th Step of glycolysis is the rapid isomerization of the triose phosphates Phase II - Payoff Phase Note: two glyceraldehydes produced per 1 glucose. 1. Step six: Oxidation and phosphorylation yielding a high-energy mixed anhydride bond. Reaction is catalyzed by Glycyeraldehyde 3-phosphate dehydrogenase. 2. Step 7: transfer of the high-energy phosphoryl group to ADP to produce ATP catalyzed Phosphoglycerate kinase 2|P a g e ©Dr. Christopher Larbie 3. Step 8: Intramolecular phosphoryl-group transfe catalyzed Phosphoglycerate kinase 4. Step 9: Dehydration to yield an energy rich enol phosphodiester catalyzed Enolase 5. Step 10: Transfer of a high energy phosphoryl group to ADP to yield ATP. Net Overall Glucose + 2ADP + 2Pi + 2NAD+ → 2Pyruvate + 2ATP + 2NADH +2H2 O + 2H+ ΔGo’ = -85 kJ/mol 9 of the ten metabolites of glycolysis are phosphorylated. Phosphorylated intermediates serve 3 functions. 1. The phosphoryl groups are ionized at physiological pH giving them a net negative electrostatic charge. Biological membranes are impermeable to charged molecules. Intermediates are held within the cell. Glucose-6-phosphate formed is negatively charged and is thus retained in the cytosol because the plasma membrane is impermeable. Also the rapid conversion of glucose to glucose -6phosphate keeps intracellular concentration of glucose low favouring facilitated diffusion of glucose into the cell. 3|P a g e ©Dr. Christopher Larbie 2. The transfer of phosphoryl groups conserves metabolic energy. The energy released in breaking the phosphoanhydride bonds of ATP is partially conserved in the formation of phosphate esters. High-energy phosphate compounds formed in glycolysis donate phosphoryl groups to ADP to form ATP. 3. The enzymes of glycolysis use the binding energy of phosphate groups to lower the activation energy and increase the specificity of the enzyme reactions. The Reactions of Glycolysis Step 1: Phosphorylation of Glucose Hexokinase transfers the γ-phosphate of ATP to the C-6 hydroxyl group of glucose to yield glucose 6-phosphate. The phosphorylation of glucose commits it to the cell. A Kinase is an enzyme that catalyzes the transfer of the terminal phosphoryl group of ATP to some acceptor nucleophile. The acceptor in the case of hexokinase is the C-6 hydroxyl group of D-glucose, D-mannose or D-fructose. Hexokinase requires Mg2+ for activity. The true substrate for this enzyme is not ATP 4- but MgATP2-. Hexokinase is the poster enzyme for the induced fit mechanism where substrate binding induces conformational changes in the enzyme. 4|P a g e ©Dr. Christopher Larbie The C-6 hydroxyl group of D-glucose has similar reactivity as water. The enzyme active site is fully accessible by water. Hexokinase discriminates against water by a factor greater than 106 . The ability to discriminate comes from the conformational changes in hexokinase when the correct substrates are bound. Only when a hexose is bound does the conformation change and the enzyme becomes active. The mechanism of the phorphoryl group transfer is the typical Sn2 nucleophilic substitution reaction. By using a chiral phosphate shown to the left, it can be shown that transfer of the γ-phosphate of ATP proceeds with inversion of configuration at the electrophilic center of the phosphorous. The kinetic mechanism of hexokinase is a random bi bi. 5|P a g e ©Dr. Christopher Larbie The complete reaction for the hexokinase reaction is: α-D-Glucose + MgATP 2- → α-D-Glucose-6-phosphate 2- + MgADP1- + H+ ΔGo’ = -16.7 kJ/mol K’eq = 850 Under cellular conditions, the first step of glycolysis is even more favourable than the standard state. Take erythrocytes for example. The steady state concentration of [ATP] = 1.85 mM, [Glucose] = 5.0 mM, [G-6-P] = 0.083 mM, and [ADP] = 0.14 mM. The free energy change under these concentrations is given by: Hexokinase is found in all cells of all organisms. Isozymes of hexokinase are found in yeast and mammals. Glucokinase is an isozyme of hexokinase sometimes called Hexokinase IV (other times called Hexokinase D). Glucokinase is found in hepato cytes (liver cells). The hexokinase isozyme found in skeletal muscle has a Km for glucose of 0.1 mM. The skeletal muscle maintains steady state concentration of glucose around 4 mM. This isozyme is allosterically inhibited by high concentrations of glucose -6phosphate. The isozyme glucokinase catalyzes the same reaction but has a very high Km for glucose of 10.0 mM. This enzyme is not allosterically regulated by high glucose -6phosphate concentrations. The high Km, means that glucokinase only becomes metabolically important when glucose levels are high. This enzyme produces glucose -6phosphate which is then stored by the liver in the form of glycogen. Glucokinase is an inducible enzyme. The amount of this enzyme present in hepatocyte cells is regulated by insulin. Step 2: Isomerization of Glucose-6-Phosphate In the second reaction of glycolysis the aldose glucose-6-phosphate is isomerized into the ketose, fructo-6-phosphate. The carbonyl is shifted from the C1 of glucose to the C2 of fructose. This reaction serves two functions. Function 1: The next step of glycolysis is phophorylation at the C1 position. The hemiacetal hydroxyl group of glucose is a poor nucleophile, while the primary hydroxyl group of fructose is a good nucleophile. Function 2: The isomerization to fructose puts the carbonyl at the C2 position which activates the C3 carbon for aldolytic cleavage. The isomerization is catalysed by the 6|P a g e ©Dr. Christopher Larbie enzyme phosphoglucoisomerase. (ΔGo’ = 1.67 kJ/mol, In erythrocytes ΔG = -2.92 kJ/mol). This enzyme functions near equilibrium under cellular concentrations. The predominant forms of glucose and fructose in solution are the ring forms shown above. Phosphoglucoisomerase must begin with the opening of the ring to interconvert G-6-P to F-6-P. 7|P a g e ©Dr. Christopher Larbie Every enzyme mechanism begins with the binding of the substrate. Step A. A general acid, presumably a lys ε-amino group catalyses the ring opening. Step B. A base presumably a carboxylate of glutamate abstracts the proton of C2 to form a cis enediolate intermediate, Step C: The proton abstracted from C2 is replaced on the C1 carbon. Ring closure then produces the products. Reaction 3: Phosphorylation of Fructose-6-Phosphate This is the second of the two priming reactions of glycolysis. The phosphorylation is catalyzed by the enzyme phosphofructokinase-1. ΔGo’ = -14.2 kJ/mol. In erythrocytes ΔG = -18.8 kJ/mol (far from equilibrium.) This reaction is the second committed step of glycolysis. Phosphofructokinase commits the glucose towards glycolysis. This is the enzyme that regulates the flux of metabolites through the pathway. It is allosterically regulated by the product. The activity of the enzyme is allosterically inhibited by ATP and Citrate (from the TCA cycle). It is activated by AMP and β-D-fructose 2,6 bisphosphate Reaction 4: Cleavage of Fructose-1,6-bisphosphate. The enzyme fructobisphosphate aldolase catalyses a reversible aldol condensation. The enzyme is commonly called aldolase. Fructose-1,6-bisphosphate is cleaved to yield two triose phosphates, glyceraldehydes 3-phosphate and dihydroxyacetone phosphate. (ΔGo’ = 23.8 kJ/mol; In erythrocytes ΔG = -0.23 kJ/mol; another near equilibrium reaction) This reaction is an aldol cleavage reaction. The bond cleave is the one between the C3 and C4 carbons. The aldol cleavage of fructose-1-6-bisphosphate results in two interconvertible C3 compounds that can enter a common degradative pathway. The enolate intermediate is stabilized by resonance. Two classes of aldolase enzymes are found in nature. Animal tissues have Class I aldolase. Class I aldolases form a covalent Shiff base between the carbonyl of the substrate and an active site lysine. This reaction has high activation energy because of the high energy carbanion character of the transition state. There two types of aldolase enzymes that catalyze this reaction. 8|P a g e ©Dr. Christopher Larbie Class I aldolases are found in plants and animals. To lower the activation energy they use covalent catalysis. An active site lysine attacks the carbonyl to form a Schiff base. The Schiff base withdraws electrons from the reaction center stabilizing the carbanion character of the transition state and lowering the activation energy. Class II aldolases are found in fungi, algae and some bacteria. They do not form a Schiff base with the substrate. Instead they lower the activating energy by polarizing the carbonyl bond and providing electrostatic stabilization of the carbanion character of the transition state with divalent metal ions such as Zn2+ or Fe2+. 9|P a g e ©Dr. Christopher Larbie Reaction 5: Triose Phosphate Isomerization. Of the two products formed in the aldolase reaction, only glyceraldehydes goes on into the second phase of glycolysis. The other triose phosphate, dihydroxyacetone phosphate must be converted into glyceraldehydes 3-phosphate by the enzyme triose phosphate isomerase. By this reaction, C1, C2 and C3 of the starting glucose become indistinguishable from C6, C5 and C4 respectively. (ΔGo’ = 7.5 kJ/mol; in erythrocytes ΔG = 2.7 kJ/mol; another reaction near equilibrium.) 10 | P a g e ©Dr. Christopher Larbie The mechanism of triose phosphate isomerase (TIM) is shown above. The mechanism proceeds with the formation of an enediol or enediolate intermediate similar to that of phosphoglucose isomerase. Glu-165 has been shown to function as a general acid/base catalyst. The pKa of Glu-165 has been perturbed to 6.5. In the first step of the reaction Glu-165 acts as a general base abstracting the proton from the C3 position of DHAP. The general acid catalyst protonating the carbonyl group is His-95. The result is the enediol intermediate. His-95 then functions as a general base abstracting the hydrogen from the hydroxyl group with Glu-165 functioning as a general acid donating its proton to the C2 carbon forming G-3-P. TIM was the first enzyme discovered to have an α/β barrel tertiary structure. A cylinder of 8 parallel β- strands is surrounded by eight α-helices. Since the discovery numerous other proteins have been found to have this tertiary structure including the glycolytic enzymes, aldolase, enolase and pyruvate kinase. Triose phosphate isomerase is an enzyme that has evolved into catalytic perfection. It catalyses the interconversions at the diffusion limit. Meaning the rate determining step is the diffusion of the substrate to the active site. Second Phases of Glycolysis In the preparatory phase of glycolysis two molecules of ATP have been invested (Step 1 and 3). The hexose chain has been cleaved into two triose phosphates. The second phase contains the last five reactions of glycolysis and is called the payoff phase. It is called the payoff phase because in these five reactions two high energy phosphate bonds are produced. These 2 high energy phosphoryl groups are transferred to ADP to generate 2 molecules of ATP. It is important to remember that two molecules of glyceraldehydes 3phosphate is generated per 1 molecule of glucose. The conversion of two molecules of 11 | P a g e ©Dr. Christopher Larbie glyceraldehyde-3-phosphate into two molecules of pyruvate is accompanied with the generation of 4 ATP molecules and 2 molecules of NADH. The net reaction for glycolysis is the production of 2 ATP. Reaction 6: Oxidation and phosphorylation of glyceraldehydes 3-phosphate The first step of the second phase of glycolysis is the oxidation and phosphorylation of glyceraldehydes 3-phosphate to form 1, 3-bisphosphoglycerate. The enzyme is glyceraldehyde 3-phosphate dehydrogenase. (ΔGo’ = 6.3 kJ/mol; In erythrocytes ΔG = -1.29 kJ/mol; another reaction near equilibrium.) 12 | P a g e ©Dr. Christopher Larbie The oxidation of an aldehyde to the carboxylic acid is an exergonic process. This enzyme uses the free energy of aldehyde oxidation to synthesize a hi gh energy acyl phosphate, 1, 3-bisphosphoglycerate. Fun facts about glyceraldehyde 3-phosphate dehydrogenase. 1. Iodoacetate, a reagent that alkylates cysteine residues, inactivates GAPDH. Iodoacetate reacts stiochiometrically with GAPDH. The confirmed presence of a carboxymethylcysteine residue demonstrates an active site cysteine residue that is essential for enzymatic activity. 2. GAPDH quantitatively transfer 2H from the C1 carbon of glyceraldehydes 3phosphate to NAD+, establishing a direct hydride transfer. 3. GAPDH catalyses 32 P exchange between inorganic phosphate and acetyl phosphate. Such isotope exchange is indicative of an acyl-enzyme intermediate. Mechanism of GAPDH 1. First the substrate G-3P binds to the enzyme. The essential cysteine residue acts as a nucleophilic and attacks the aldehyde carbonyl to form a thiohemiacetal intermediate. 2. Next the thiohemiacetal transfer a hydride (2e - + H+) to the active site bound NAD+ to form NADH and a thioester intermediate. This NADH molecule dissociates from the active site and another NAD + molecule is bound. The enzyme binds a molecule of inorganic phosphate which is the nucleophile that attacks the thioester To regenerate the active enzyme’s sulfhydryl and form 1, 3bisphosphoglycerate. 13 | P a g e ©Dr. Christopher Larbie Reaction 7: Phosphoryl transfer from 1,3-bisphosphoglycerate The enzyme phosphoglycerate kinase catalyses the transfer of the high energy acyl phosphate to ADP. The result is the synthesis of two molecules of ATP. This enzyme pays off the ATP debt of the first phase of glycolysis. (ΔGo’ = -18.9 kJ/mol; In erythrocytes ΔG = 0.1 kJ/mol; another reaction near equilibrium.) Substrate level phosphorylation occurs when ADP is phosphorylated to form ATP at the expense of the substrate which is 1,3-bisphosphoglycerate in this case. 14 | P a g e ©Dr. Christopher Larbie Reaction 8: Conversion of 3-phosphoglycerate into 2-phosphateglycerate The enzyme phosphoglycerate mutase catalyses the reversible shift of the phosphate ester between C2 and C3 of glycerate. A mutase is an enzyme that catalyses the migration of a functional group within the substrate molecule. Mg2+ is required for enzymatic activity. (ΔGo’ = 4.4 kJ/mol; In erythrocytes ΔG = 0.83 kJ/mol; another reaction near equilibrium.) This reaction is more complicated than it looks. Phosphoglycerate mutase has a covalent phosphoenzyme intermediate and requires 2,3-bisphosphoglycerate as a cofactor. The phosphoenzyme has a phosphoryl group covalently bound to an active site histidine residue. The phosphoenzyme binds 3-phosphoglycerate and transfer the phosphoryl group from the histidine to the C2 position of 3-phosphoglycerate to form 2,3-bisphosphoglycerate. 2,3-bisphosphoglycerate then transfers the C3 phosphoryl group to the active site histidine residue to regenerate the active phosphoenzyme and produce 2phosphoglycerate. Once in every 100 turnovers, the intermediate 2,3-bisphosphoglycerate escapes from the enzyme, leaving an inactive dephosphorylated enzyme. The unphosphorylated enzyme binds 2,3- bisphosphoglycerate and transfers the C3 phosphoryl group to the histidine reactivating the enzyme and producing 2-phosphoglycerate. For this reason, the enzyme requires a small amount of 2,3-BPG as a cofactor for maximal activity. 15 | P a g e ©Dr. Christopher Larbie Reaction 9: Dehydration of 2-phosphoglycerate Enolase is the enzyme that catalyses the reversible elimination reaction of water to convert 2-phosphoglycerate into phosphoenol pyruvate. Mg2+ required for enzymatic activity. (ΔGo’ = 7.5 kJ/mol; In erythrocytes ΔG = 1.1 kJ/mol; Another reaction near equilibrium) 16 | P a g e ©Dr. Christopher Larbie Enolase dehydrates the substrate to form a high energy phosphoenol. The standard change in free energy for the hydrolysis of phosphoenolpyruvate is a −62 kJ/mol. This enzyme is strongly inhibited by fluoride in the presence of phosphate. Fluoride reacts with phosphate to form fluorophosphates (FPO 3 2-) which complexes with the Mg2+ ion located in the active site of the enzyme. Reaction 10: Transfer of the phosphoryl group of phosphoenolpyruvate. The last step of glycolysis is the transfer of the phosphoryl group of phosphoenol pyruvate to ADP to form ATP and pyruvate. The enzyme that catalyses this reaction is pyruvate kinase. This enzyme binds MgADP or MgATP 2- i.e. Mg2+ required for enzymatic activity. (ΔGo’ = -31.4 kJ/mol; In erythrocytes ΔG = -23.0 kJ/mol; Far from equilibrium.) Since 2 PEP are formed for every glucose molecule that enters the Glycolytic pathway, 2 ATP molecules are formed in this step. The ATP debt generated during phase 1 of glycolysis was paid by the formation of ATP by the substrate level phosphorylation of ADP by 1,3-bisphosphoglycerate. The 2 ATP molecules produced here the payoff for glycolysis. The large negative ΔG of this reaction makes this enzyme a target for regulation. Pyruvate kinase has binding sites for a number of allosteric effectors. ↑AMP ↓ATP ↑Fructose 1,6-bisphosphate ↓Acetyl-CoA ↓Alanine 17 | P a g e ©Dr. Christopher Larbie In addition to the allosteric effectors, pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a cAMP-dependent protein kinase which transfers the γ- phosphate of ATP to the pyruvate kinase. The phosphorylated pyruvate kinase is more strongly inhibited by ATP and alanine. The Km for PEP for the phosphorylated enzyme is also increased to the point that at the steady state physiological concentrations of PEP, the enzyme is completely inactive. Summary of Glycolysis Fates of Pyruvate The two molecules of pyruvate and 2 molecules of NADH produced from one molecule of glucose during glycolysis have three possible metabolic fates. Under aerobic conditions, pyruvate is oxidized, with the loss of CO 2 to produce acetyl-CoA and NADH. Acetyl- CoA then enters the citric acid cycle where it is completely oxidized into CO 2 and H2 O. Under aerobic conditions the NADH produced from glycolysis and 18 | P a g e ©Dr. Christopher Larbie the citric acid cycle are reoxidized into NAD + in the mitochondrial electron transport chain. Under anaerobic conditions NADH accumulates as a product of GAPDH catalysed sixth step of glycolysis. NAD+ is required as a substrate for GADPH and soon becomes limiting. In order to maintain production of ATP via the glycolytic pathway under anaerobic conditions, NAD+ has to be regenerated from NADH. There are two possible anaerobic pathways. 1. Homolactic acid fermentation Under anaerobic conditions, such as when muscle cells are vigorously working, pyruvate is reduced to lactic acid to regenerate NAD+. This process is called homolactic fermentation. (For lactate dehydrogenase, ΔGo’ = −25.1 kJ/mol; In erythrocytes ΔG = −14.8 kJ/mol). Speaking of erythrocytes, red blood cells do not have mitochondrion. They convert glucose into 2 molecules of lactate even under aerobic conditions. Other tissues (retina, brain) convert glucose into two molecules of lactate under anaerobic conditions. 19 | P a g e ©Dr. Christopher Larbie Lactate dehydrogenase, LDH catalyses the reduction of pyruvate to lactate with absolute stereospecificity. The Pro-R hydrogen of NADH is transferred to the pyruvate to form L-lactate. The mechanism of LDH is shown to the below. The pro-R hydride is transferred from NADH to C2 of pyruvate with the concomitant transfer of a proton from the imidazolium His-195. 20 | P a g e ©Dr. Christopher Larbie 2. Alcoholic fermentation Under anaerobic conditions some plant tissues, invertebrates, protoctists, and microorganisms (yeast) regenerate NAD+ by alcoholic fermentation. Pyruvate and NADH are converted into ethanol, CO 2 and NAD+. Yeast under anaerobic conditions regenerates NAD+ by alcoholic fermentation. This is an important reaction in making beer, wine and spirits. In addition the CO 2 produced by alcoholic fermentation leavens bread. The conversion of pyruvate into ethanol is a two-step process. The first step involves the decarboxylation of pyruvate by the enzyme pyruvate decarboxylase. This enzyme requires the coenzyme thiamine pyrophosphate, TPP. This cofactor is bound tightly by the enzyme. Thiamine pyrophosphate functions as an electron sink to stabilize the build-up of negative charge of the carbonyl carbon atom in the transition state. 21 | P a g e ©Dr. Christopher Larbie Mechanism of pyruvate carboxylase 22 | P a g e ©Dr. Christopher Larbie The next step of alcoholic fermentation is the reduction of acetaldehyde by NADH to produce ethanol and NAD+. The enzyme that does this reduction is alcohol dehyrogenase, ADH. Yeast alcohol dehydrogenase contains an active site Zn2+ ion. This zinc polarizes the carbonyl bond of acetaldehyde and to stabilize the negative charge that is built up in the transition state. ADH is stereospecific; it transfers the pro-R hydride of NADH to the pro-R position of ethanol. 23 | P a g e ©Dr. Christopher Larbie In the Mammalian liver, there is ADH that catalyses the opposite reaction; the oxidation of ethanol in to acetaldehyde. This enzyme has two Zn2+ ions in the active site although only one of the Zincs participates directly in catalysis through the same general mechanism. Thus excess consumption of ethanol can lead to the generation of oxygen radicals, leading to the peroxidation of lipids and alcoholic fatty liver diseases. Energetics of Homolactic acid Fermentation Effeciency of Homolactic acid Fermentation. 2 ATP’s produced 61 kJ/mol. Standard free energy change in converting glucose into two molecules of lactate, ΔGo’ = -196.0 kJ/mol. Efficiency = 100 X 61/196 = 31% Under the steady state concentrations of the cell the efficiency is greater than 50%. 24 | P a g e ©Dr. Christopher Larbie Energetics of alcoholic fermentation Efficiency = 100 X 61/235 = 26% Under the steady state concentrations of the cell the efficiency is greater than 50%. Glycolysis is used for rapid ATP production The rate of ATP formation in anaerobic glycolysis is 100 times faster than ATP production by oxidative phosphorylation in the mitochondria (aerobic). When tissues are rapidly consuming ATP, they generate it almost entirely by anaerobic glycolysis. There two type of muscle fibres; Fast twitch and Slow twitch. Fast-twitch fibres are capable of short burst of rapid activity. The cells that compose these fibres are almost completely devoid of mitochondria. They generate nearly all of their energy by anaerobic glycolysis. They are easily identifiable as white fibres. The flight muscles of the land loving birds such as chickens and turkeys are used only for short burst of flight to escape danger. Their flight muscles are composed mainly of fast twitch fibres that give them white breast meat. Sprinters muscles are mainly fast-twitch fibres. Slow-twitch fibres contract slowly and steadily. They are rich in mitochondria and obtain most of their energy by oxidative phosphorylation. The high concentration of mitochondria gives these muscle fibres a red colour to the haem containing cytochromes. The flight muscles of birds are an illustrative example. The flight muscles of migratory birds such as ducks and geese are rich in slow twitch fibres and therefore these birds have dark breast meat. Marathon runners mainly slow-twitch fibres. Glucose Transporters The lipid bilayer of the cellular membrane is an effective barrier to the movement of glucose across it. That would be a problem if there were not a means of transporting glucose into cells. Remember in multicellular organisms that glucose is made in specialized organs, such as the liver and is transported by the bloodstream to desired tissues. The job of moving glucose into cells is undertaken by specialized glucose transporters (GLUT proteins). The table below shows several glucose transporters and their tissue locations. The most common of these are GLUT1 and GLUT3, which are found in almost all mammalian cells. 25 | P a g e ©Dr. Christopher Larbie Cancer and Glycolysis Cancer cells have faster metabolic rates than normal cells and consequently must uptake and metabolize glucose faster than normal cells. Rapid metabolism leads to hypoxia (lack of oxygen compared to the need for it). Cancer cells respond to hypoxia by activating a transcription factor called Hypoxia-Inducible Transcription Factor (HIF1). HIF-1 acts as a transcription factor to increase the transcription and translation of most glycolytic enzymes and GLUT1 and GLUT3. Thus, these cells have both an increased supply of glucose and increased amounts of glycolyti c enzymes to break it down. Remember that when oxygen is limiting, glycolysis is much less efficient, so to keep the energy levels high, cells must run more cycles of glycolysis. This is made possible by having increased transport and increased numbers of glycolytic enzymes. 26 | P a g e ©Dr. Christopher Larbie REGULATION OF GLYCOLYSIS Regulatory mechanisms for glycolysis include 1. Allosteric regulation 2. Hormonal control (via enzyme phosphorylation) 3. Substrate level control 4. Covalent modification (phosphorylation via the kinase cascade Because the principal function of glycolysis is to produce ATP, it must be regulated so that ATP is generated only when needed. The enzyme which controls the flux of metabolites through the glycolytic pathway is phosphofructokinase (PFK -1). PFK-1 is an allosteric enzyme that occupies the key regulatory position for glycolysis. PFK -1 has a tetrameric enzyme composed of four identical subunits. Like other allosteric proteins (haemoglobin) and enzymes (ATCase) the binding of allosteric effectors and substrates is communicated to each of the active sites. Quaternary changes are concerted and preserve the symmetry of the tetramer. PFK -1 has two sets of alternative interactions between subunits which are stabilized by hydrogen bonds and electrostatic interactions. The two set of conformations are called the T and R states. These two conformational states are in equilibrium: T ↔ R I. ATP feedback inhibition. The function of the glycolytic pathway is to generate ATP. ATP is both a substrate and an allosteric inactivator. The enzyme has two binding sites for ATP. One is the substrate binding site and the other one is an inhibitory site. The PFK-1 substrate binding site binds ATP equally well in both the T and R states. The inhibitory ATP binding site only binds ATP when the enzyme is in the T conformation. The other substrate fructose-6-phosphate binds only to the R state. High concentrations 27 | P a g e ©Dr. Christopher Larbie of ATP shift the equilibrium towards the T conformation which decreases the affinity of the enzyme for F-6-P. II. AMP reverses inhibition. AMP reverses the inhibition due to high concentrations of ATP. AMP binds preferentially to the R state of PFK. This is important; the concentration of ATP drops only 10 % during vigorous exercising. AMP concentration levels in the cell can rise dramatically due to the enzymatic activity of adenylate kinase. The steady state concentration of ATP in the cell is 10 times greater than the concentration of ADP, and 50 times the concentration of AMP. As a result of the activity of adenylate kinase, a 10% decrease in the concentration of ATP results in 400 % increase in the concentration of AMP. III Other allosteric effectors of PFK-1 ADP is another allosteric effector of PFK-1. ADP like AMP reverses the inhibitory effects of ATP and is therefore considered an allosteric activator. The activity of PFK -1 is dependent on the ATP, ADP and AMP concentrations which are all functions of the cellular energy status. Other regulators of PFK activity 1. Under aerobic conditions, the pyruvate formed by glycolysis is fed into the citric acid cycle where it is completely oxidized into CO 2 and H2 O. Citrate is a metabolite of the citric acid cycle. When the citric acid cycle is saturated, glycolysis needs to be slowed down. When the citric acid cycle is saturated, the citrate concentration in the cytosol increases. Citrate binds preferentially to the T state of PFK-1. Thus high concentrations of citrate inactivate the enzyme. 28 | P a g e ©Dr. Christopher Larbie 2. PFK-1 is also regulated by β-D-fructose 2-6-bisphosphate which is a potent allosteric activator of the enzyme. β-D-fructose 2-6-bisphosphate binds to the Rstate of the enzyme and increases the affinity of the enzyme for fructose-6phosphate. β-D-fructose 2-6-bisphosphate also decreases the inhibitory effects of ATP 3. Regulation in Muscle by negative feedback effect and feed forward stimulation. 29 | P a g e ©Dr. Christopher Larbie Summary of Regulation of Glycolysis Hormonal Regulation Glycolysis is also regulated by the peptide hormones glucagon and insulin. Glucagon, released by pancreatic α-cells when blood glucose is low, activates the phosphatase function of PFK-2, thereby reducing the level of fructose-2,6-bisphosphate in the cell. As a result, PFK-1 activity and flux through glycolysis are decreased. In liver, glucagon also inactivates pyruvate kinase. Glucagon’s effects, triggered by binding to its receptor on target cell surfaces, are mediated by cyclic AMP. Cyclic AMP (cAMP) is synthesized from ATP in a reaction catalysed by adenylate cyclase, a plasma membrane protein. Once synthesized, cAMP binds to and activates protein kinase A (PKA). PKA then initiates a signal cascade of phosphorylation/dephosphorylation reactions that alter the activities of a diverse set of enzymes and transcription factors. Transcription factors are proteins that regulate or initiate RNA synthesis by binding to specific DNA sequences called response elements. Insulin is a peptide hormone released from pancreatic β-cells when blood glucose levels are high. The effects of insulin on glycolysis include activation of the kinase function of PFK-2, which increases the level of fructose-2,6-bisphosphate in the cell, in turn increasing glycolytic flux. In cells containing insulin-sensitive glucose transporters (muscle and adipose tissue but not liver or brain) insulin promotes the translocation of glucose transporters to the cell surface. When insulin binds to its cell-surface receptor, 30 | P a g e ©Dr. Christopher Larbie the receptor protein undergoes several autophosphorylation reactions, which trigger numerous intracellular signal cascades that involve phosphorylation and dephosphorylation of target enzymes and transcription factors. Many of insulin’s effects on gene expression are mediated by the transcription factor SREBP1c, a sterol regulatory element binding protein. As a result of SREBP1c activation, there is increased synthesis of glucokinase and pyruvate kinase. 31 | P a g e ©Dr. Christopher Larbie METABOLISM OF HEXOSES OTHER THAN GLUCOSE I. Fructose Sucrose is a major fuel source of our diets. Sucrose is a disaccharide of fructose and glucose. There are two pathways for the metabolism of fructose. The first and simplest pathway for fructose catabolism occurs in the muscle cells where fructose is a substrate for hexokinase which produces fructose-6-phosphate, a substrate for PFK-1. The liver, however, contains little hexokinase. Instead it contains glucokinase which specifically phosphorylates glucose. The liver catabolizes fructose through a pathway that involves six enzymes. 32 | P a g e ©Dr. Christopher Larbie Step 1: In the liver the first step of fructose catabolism is the phosphorylation of fructose by fructokinase to form fructose-1-phosphate. Note that neither hexokinase or phosphofructokinase can phosphorylate fructose-1-phosphate into fructose-1,6bisphosphate. Step 2: In the liver we find a class I aldolase which is an isozyme of fructose bisphosphate aldolase (aldolase Type A). The Type A aldolase is specific for the substrate fructose-1,6bisphosphate. The isozyme of aldolase found in the liver is called a Type B aldolase. It can utilize fructose-1-phosphate as well as fructose-1,6-bisphosphate as a substrate. So the Type B aldolase found in the liver catalyses the aldolytic cleavage of fructose-1phosphate into glyceraldehydes and dihydroxyacetone phosphate. Step 3: The dihydroxyacetone phosphate produced can be converted into glyceraldehyde 3 phosphate by the reaction catalyzed by triose phosphate isomerase. The other product of the aldolytic cleavage, glyceraldehydes can be directly phosphorylated by glyceraldehyde kinase to form glyceraldehydes 3-phosphate. 33 | P a g e ©Dr. Christopher Larbie Step 4: There is an alternative pathway where the glyceraldehydes is reduced by alcohol dehydrogenase into glycerol. Glycerol is then phosphorylated by glycerol kinase to form glycerol-3-phosphate. The glycerol 3-phosphate is then oxidized by glycerol phosphate dehydrogenase into dihydroxyacetone phosphate which of course is converted into glyceraldehydes 3-phosphate by triose phosphate isomerase. II Mannose Mannose is the C2 epimer of glucose. Mannose is a substrate for hexokinase which converts it into mannose 6-phosphate. An enzyme similar to phosphoglucose isomerase, phosphomannose isomerase isomerizes mannose 6- phosphate into fructose-6-phosphate. Fructose 6-phosphate is the substrate for PFK-1. III Galactose Galactose is the C4 epimer of glucose. The entry of galactose into glycolysis begins with the phosphorylation of galactose by galactokinase to form galactose 1-phosphate. Galactose 1-phosphate is then converted into UDP-galactose by the enzyme galactose-1phosphate uridylyltransferase with the concurrent formation of glucose-1-phosphate from UDP-glucose. 34 | P a g e ©Dr. Christopher Larbie This enzyme has a ping pong kinetic mechanism. UDP-glucose binds to the enzyme first. A covalent enzyme-UMP intermediate is formed liberating glucose-1-phosphate which dissociates from the enzyme. Galactose-1- phosphate then binds and reacts with the covalent UMP-enzyme intermediate to form UDP-galactose which then dissociates from the enzyme. Galactosemia is the genetic disease caused by the inability of the individual to convert galactose into glucose. The symptoms of it are mental retardation, liver damage and cataracts (by the conversion of galactose to galactitol). This genetic disease is caused by a deficiency of the galactose-1-phosphate uridylyltransferase enzyme. Galactosemia is treated by a galactose free diet which reverses all of the symptoms except for the mental retardation. 35 | P a g e ©Dr. Christopher Larbie The glucose 1-phosphate formed in the first step of this reaction is converted into glucose-6-phosphate by the enzyme phosphoglucomutase. The UDP-galactose is converted into UDP-glucose by the enzyme UDP-glucose-4-epimerase. This enzyme has an active site NAD+ bound and functions through the sequential oxidation and reduction of the C4 atom. The net result of UDP-glucose uridylyltransferase and UDP-glucose-4-epimerase is the conversion of galactose-1-phosphate into glucose-1-phosphate. IV Lactose: Lactose (a disaccharide of glucose and galactose) is a problem in metabolism for many adults, due to deficiency of the enzyme lactase. Side effects arise from growth of colon bacteria on the undigested lactose, creating gas and diarrhoea. Galactose too can be a metabolic problem for some people, due to the deficiency of the enzyme galactose-1-phosphate uridyl transferas. In these people, cataracts form, the liver can enlarge, and jaundice is common, upon drinking of milk. Infants with the syndrome may die of it. Cataracts arise due to conversion of the excess galactose to galactitol in the lens of the eye, encouraging the diffusion of water into the lens and the start of cataract formation. 36 | P a g e ©Dr. Christopher Larbie