Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Soc 460 7 Dr Mresfeld Sprmg 2008 Glycolysis 2 Supplemental reading Key Concepts Regulation of the Glycolytic Pathway Glucokinase is a molecular sensor ofhigh glucose levels Allosteric control of ph ospho 39uctokin ase activity Supply and demand of glycolytic intermediates Metabolic Fate of Pyruvate carter tamer KEY CONCEPT QuEsnoNsm GchoLvsIs How do substrate availability and enzyme activity levels control glycolytic flux Why is muscle lactate dehydrogenase activity required for short bursts of intense exercise BiochemicalAQQications of Glycolzs drseas arner Carrel nanmi Inherited genetic defects in metabolic enzymes can give rise to human diseases that are caused by decreased levels of enzyme or altered enzyme activities complete loss ofthe enzyme is olten embryonic lethal Enzyme de ciencies in gluckokinase lactate dehydrogenase and 39uctose1 P aldolase all lead to metabolic diseases REGULATION OF THE GLYCOLYTIC PATHWAY Irreversible reactions in metabolic R we 1 pathways are called rateli ng steps J r because the level of enzyme activity can be low even when substrate levels are Hmkinase L r mumse5pmphame r high Ratelimiting enzymes in metabolic Girizwmol L Girswmol pathways serve as regulated valves that are opened or closed in response to 3 cellular conditions As illustrated in gure 1 reversible steps in glycolysis and Fhospholrunukrnase gluconeogenesis operate in both pathways A W m whereas irreversible steps have actual changes in 39ee energies AG that are highly negative and require pathway speci c enzymes Glucokinase is a molecular sensor of high glucose levels 3 Four hexokinase genes have been identi ed in humans hexokinase V all ofwhich are capable of converting glucose y unusel briphnsphalzse m e 55 mm K j 1 FLUCONEOGENEs z N to glucose6 P at the expense of ATP V P hydrolysis step 1 of glycolysis We have PyruvalekinaseL JAPEPquot39L k5 m already described one ofthese 5 WWW j 1 Pymmcmmoxylasc hexokinase l reaction 1 which has a high af nity for substrate Km for glucose is V 1 of9 pages Bioc 460 Dr Miesfeld Spring 2008 O lmM is expressed in all tissues phosphorylates a variety of hexose sugars and is inhibited by the product ofthe reaction glucose6P In contrast hexokinase IV also known as glucokinase has a low affinity for substrate Km for glucose is 10mM is highly specific for glucose is expressed primarily in liver and pancreatic cells and is not inhibited by glucose6P This difference in tissue expression and glucose affinity between hexokinase and glucokinase plays an important role in controlling blood glucose levels which ultimately controls rates ofglycolytic flux in all cells by limiting substrate availability As suggested by the different Km values of hexokinase and glucokinase for glucose substrate saturation cunes for these two enzymes look markedly different as shown in figure 2 Since blood glucose levels are maintained around 5mM significant levels of glucose phosphorylation by glucokinase only occur under conditions of high glucose such as after consuming a carbohydraterich meal Moreover since glucokinase is not inhibited by glucose6P it is able to continue functioning even if flux through glycolysis cannot keep up with product formation The role of glucokinase in liver cells is to trap the extra glucose that is available from the diet so that it can be stored as glycogen for an energy source later By being active in liver cells only when glucose concentrations exceed normal limits gt5mM glucokinase ensures that the liver is the Figure 3 major sink for dietary glucose l StimulationowWE Glucose Figure 2 Normal blood glucose concentration 1 0 Hexokinase l Glucokinase Reaction Rate 5 Vmax Km 10AM 274 5 871012 i4 1 6 1 8 2390 Glucose concentration mM Insulin K I I nsum I 1 395 si nalin 9 9 II at a i39 Pmtem lucokinase and at the same time is able to anymeeve5 efficiently remove glucose from l Glucose 1 gt3 Ca2 stimulates insulin the blood to help restore ATP 1 vesicles to fuse with normal blood glucose ADP G quot kquotquot 59 K1 plasma membrane concentrations Another 4 39J39 a important function of Glucose 6P C 2 glucokinase is to act as a I Glycolysis 39 39 an a glucose sensor in pancreatic ATP inhibits D a 3 cells where glucokinase Pymvate K channel 0 Activation of enzyme levels are activated by v z VO thge gatEd increased glucose import i Energy ATP K C Channe K n so mediated by glucose j 0 We transporter proteins GLUT proteins As shown in figure g when the concentration of glucose in the blood is elevated glucose import into Pancreatic 5 cell Membrane a depolarization 70 pg d Ca2 2 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 pancreatic 5 cells leads to higher glucokinase enzyme levels resulting in increased flux through glycolysis and net ATP synthesis This increase in ATP levels causes inhibition of ATPsensitive K channels membrane depolarization and activation of voltagegated Ca2 channels Elevated levels of intracellular Ca2 triggers fusion of insulincontaining vesicles with the plasma membrane and subsequent release of insulin into the blood This intracellular signaling pathway links glucose uptake in pancreatic 3 cells with insulin release The importance of pancreatic glucokinase in insulin secretion was confirmed using transgenic mice in which the glucokinase gene was specifically deleted in pancreatic 5 cells These glucokinasedeficient mice were defected in glucosestimulated insulin secretion and became hyperglycemic eventually developing diabetic symptoms due to chronic elevated blood glucose Well over 100 mutations in the human glucokinase gene have been identified and it is now known that a form of type II diabetes in humans called maturityonset diabetes ofthe young MODY2 is caused by defects in pancreatic glucokinase activity Allosteric control of phosphofructokinase activity Of all the enzymes in glycolysis phosphofructokinase is the best characterized because of its vital role in controlling flux through the pathway There are actually two phosphofructokinase isozymes distinct genes that encode proteins with similar functions phosophofructokinase1 PFK1 which catalyzes reaction 3 in glycolysis and phosphofructokinase2 PFK2 a bifunctional enzyme that catalyzes the synthesis of fructose26bisophosphate F26 BP a potent allosteric regulator of PFK1 activity discussed in lecture 35 The PFK1 reaction in glycolysis is irreversible and functions as one of three metabolic valves that controls flux through the pathway the other two are the hexokinase and pyruvate kinase reactions Figure 4 illustrates that AMP ADP and F26 BP are activators of PFK1 activity and ATP and citrate function as inhibitors HEM F26BP AMP Citrate ADP ATP Fructose6 P ATP Fructose 16BP ADP Phosphofrurtokinasel PFK1 is an allosteric enzyme that exists as a tetramer HEM a dimer of dimers in either of two conformations the inactive T state or active R state analogous to the yan39zm alBlgFK flim ng39 hemoglobintetramer The equilibrium between T and R y 39 39 39 3 cm 39 39 1 states in a cell is controlled by allosteric effector F4164 molecules which bind to a regulatory site outside of the substrate binding pocket ATP and citrate are negative effector molecules of PFK1 which stabilize the T state whereas AMP ADP and F26 BP are positive effector molecules that stabilize the R state Figure 5 shows how the allosteric regulators ATP AMP and F26 BP alter the PFK1 reaction rate as a function of substrate concentration fructose6P PFK Enzyme Activity 0 Fructosee6P W gt 3 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 6 shows the molecular structure of PFK 1 as a monomer with reaction products fructose 16BP and ADP in the active site and as a dimer in the active R conformation with ADP bound to aosteric effector site Note that the aosteric effector site is far removed from the catalytic site and in fact maps to the interface between two PFK 1 subunits Figure 6 Phosphofructokinasel dimer in the R state PhosphofructokinaseJ monomer ADP in the aosteric site Products in the enzyme active site Studies have shown that ATP binds with equal affinity to the catalytic site regardless of the T or R state conformation of PFK 1 However ATP binding to the aosteric effector site is highest when the protein is in the T state which functions to decrease fructose6P binding to the catalytic site As illustrated in figure 7 AMP binding to the aosteric effector site serves to stabilize the R state and thereby stimulates the production of fructose1 6BP by ATPmediated phosphoryl transfer This equilibrium shift between the T or R states is modulated by the energy charge of the cell such that high ATP concentrations high energy charge increase the pool of PFK 1 molecules in the T state whereas high AMP concentrations shifts the pool to more PFK1 molecules in the direction of the R state Figure 7 High energy charge in the cell Low energy charge in the cell ATP AMP V NoFr rP binding ATP 4 5 ATP ATP N f ATP N N F7671 biiiding x a X ATP ATP AMP T State R State inactive active 4 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 Supply and demand of glycolytic intermediates We have so far described the starting point for glycolysis as glucose import into the cell from the circulatory system via GLUT transport proteins followed by hexokinase or glucokinase catalyzed phosphorylation to produce glucoseGP Glucose can also be derived from glycogen which we already mentioned is the storage form of glucose polymers in liver and muscle cells When glucose is needed as energy source for muscle cells undergoing contraction glycogen is degraded by the enzyme glycogen phosphorylase to produce glucose1P Plants can t make glycogen but they do store glucose as large polymeric molecules in starch granules Animals that eat plants such as ourselves break down dietary starch into the disaccharide maltose by an enzyme in saliva called aamylase As shown in figure 8 maltose is then cleaved by the enzyme maltase in the intestine to produce two molecules of glucose Other dietary sources of carbohydrates are the disaccharides sucrose and lactose which are cleaved by the hydrolytic enzymes sucrase and lactase respectively Figure 8 Maltese 77gt H20 Glucose c v r filly H GLIJILO II L Sucrase quot394 Di 7 Glucose H U u a l I H5 Fructose 7Glucose r H TTI 7 4 5H Galactose C I 8 KC Lipase Quid w Triglyceride a quot3quot 5M 3 CH 0 l4 A 31m anquot Eau Fany c Fatty Fauy and 39lC l39ll39f acidZ and andL Glycerd 39 into the monosaccharides glucose fructose and galactose Sucrose is common table sugar and fructose is present at high levels in many types of fruits and vegetables Glycerol is a three carbon metabolite that is a component of triglycerides which contain three covalently linked fatty acid molecules A class of hydrolytic enzymes known as lipases cleave off the fatty acids from the triglyceride molecule to release glycerol which can enter the glycolytic pathway The fatty acid components oftriglycerides are metabolized in mitochondria by the fatty acid oxidation pathway to produce acetyl CoA The conversion of lactose sometimes called milk sugar to galactose and glucose by the enzyme lactase may be familiar to you if you are lactoseintolerant lactosesensitive Individuals with this condition experience considerable discomfort from associated intestinal symptoms excessive flatulence and diarrhea when lactosecontaining foods such as dairy products are eaten The human gene for lactase is expressed at high levels in infants to digest lactose in breast milk however lactase expression normally declines in adults with the notable 5 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 exception of individuals of Scandinavian descent The intestinal symptoms of lactose intolerance are caused by the activity of naturally occurring anaerobic bacteria in the human intestine from the genus Lactobacilus which ferment the undigested lactose to lactate producing hydrogen H2 and methane CH4 gases as side products Diarrhea becomes a problem if the amount of unhydrolyzed lactose is so high that it osmotically increases water flow into the intestine The simplest way to prevent these symptoms is to not eat food products containing lactose for example by eating soy milk Figure 9 products rather than dairy 7 W products Fortunately Sucrose l M E 3E E33352 biotechnology has provided a way Sumse Maltase Lactose to have your ice cream and eat it too through the industrial production of purified lactase enzyme see biochemical iGalactose Glucose 5 Galacrokinase 39 H k l ADP exo mase ADP Galactose l P Fructokinase HEXOkinClSe applications on page 1 of the liver muscle Glucose 643 EUDPglucoseltgt lecture notes ADP 1 a ADP UDPgalactose Fructose galactose and Glucose1P glycerol enter the glycolytic Fructose 1P Fructose 5 pathway through a variety of A pit7 routes many of which require Fzzcrlose IBP ADP additional enzymatic reactions as a 0 15 Fructose16BP shown in figure 9 Glycerol for l Glycerol3P example enters glycolysis through Glyceraldehyde 39dehydmg lwse I 3P a two step reaction requiring the DHAP 39 39 NADLQ NAB ycero 39 enzymes glycerol kinase and t ADP r ADP Gwem glycerol 3phosphate GAP kinase dehydrogenase to form the glycolytic intermediate 1393Bisphosphoglycerate dihydroxyacetoneP DHAP Fattyacldx Tupasa Some metabolites enter glycolysis L Fatty acid 4 LipaseZ through a single phosphorylation f 39 reaction such as fructose in w muscle cells which is converted to Pyruvate Triglycerides Fatty acid UPGSQ 7 fructose6P by the enzyme Igtco2 hexokinase Fructose AcetyICoA metabolism in liver cells however is more complicated in that dietary fructose is first converted to fructose1P by the enzyme fructokinase Fructose1P is cleaved by fructose1P aldolase to generate dihydroxyacetoneP and glyceraldehyde DihydroxyacetoneP is isomerized to glyceraldehyde3P by the glycolytic enzyme trios phosphate isomerase and glyceraldehyde is phosphorylated by triose kinase to produce glyceraldehyde3P Fructose metabolism uses the same number of invested ATP molecules as does glucose in both muscle 1 ATP and liver cells 2 ATP and is a good source of dietary carbohydrate In fact high fructose corn syrup is the most common added sweetener to processed foods However for individuals with a genetic disease called fructose intolerance fructose in the diet can be extremely toxic Fructose intolerance is due to deficiencies in fructose1P aldolase also called aldolase B People with fructose intolerance cannot eat foods containing fructose because it leads to the buildup of fructose1P which has no alternate metabolic fates in the absence of fructose1 P aldolase Since Pi turnover in cells is required to continually synthesize large amounts of ATP by 6 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 oxidative phosphorylation the accumulation of fructose1P acts as a P sink by tying up available phosphate in the liver that would normally be recycled by ATP hydrolysis Under these conditions liver cells are quickly depleted of ATP causing ATPdependent cation membrane pumps to shut down resulting in cell lysis and liver damage Glycolytic intermediates also serve important roles in anabolic pathways by providing carbon skeletons for biosynthesis Besides playing a central role in gluconeogenesis numerous glycolytic intermediates are used in a variety of pathways including amino acid biosynthesis and in the formation glycolipids and glycoproteins as illustrated in figure E This figure also shows that glycolytic intermediates generate two important regulatory molecules 23bisphosphoglycerate BPG and fructose26bisphosphate F 26BP through onestep phosphorylation reactions BPG is important in hemoglobin function and F26 BP is an allosteric regulator of phosphofructokinase1 METABOLIC FATE OF PYRUVATE The end product of glycolysis pyruvate is metabolized in one of three ways depending on the organism and the availability of oxygen as illustrated in figure 11 First under aerobic conditions the majority of pyruvate is metabolized Figure 10 km Glycogen Nucleotides 171 35332 mm 1 NADP 139 Glycolipids NADPH quot C z mlnosu ars Pemose Phosphate iFrUCtosei6iP I Am Pathway hosphofrucrokinase J P quotI Fructosel 6 BP i DHAP a 1343 v u GlyceraldehydeSPL g Triglyceridm e l3Bisphosphoglycerate x I b 3 Phosphoglycerate a F J 2phosphoglycerate Phosphoenolpyruvate Tyrosine T lwtajej u a Alanine I Glyccprcteins ructoslerBP F726 BP Regulation of oxygen transport F 9 23 Bisphosphoglycerate I L BPG 7 vv we a Pyrimidines Asparagine t in the mitochondria to acetyl CoA and ultimately to 002 and H20 which are the products ofthe citrate cycle and electron transport chain Aerobic metabolism in the mitochondrial matrix is responsible for the majority of ATP synthesis in cells that depend on the oxidation of metabolic fuel for energy conversion plants obtain energy for ATP synthesis from sunlight Second under anaerobic conditions such as occurs in muscle cells during strenuous exercise or in erythrocytes which lack mitochondria pyruvate is converted to lactate the ionized form of lactic acid by the enzyme lactate dehydrogenase A variety of microorganisms also convert pyruvate to lactate under anaerobic conditions for example Lactobacillus bulgaricus a strain of bacteria used in the dairy industry to produce foods such as yogurt and cheese The third fate of pyruvate occurs in microorganisms such as yeast which utilize alcoholic fermentation to convert pyruvate to 002 and ethanol using the enzymes pyruvate decarboxylase and alcohol dehydrogenase respectively 7 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 11 Electron Transport and Oxidative Phosphoryarian Aerobic Metabolism As shown in figure 11 these three pyruvate pathways are responsible for regenerating the coenzyme NAD required to maintain flux through the glyceraldehyde3 P dehydrogenase reaction in glycolysis This metabolic requirement for NAD in glycolysis can be seen in individuals who have defects in the enzyme lactate dehydrogenase a genetic disease called lactate dehydrogenase deficiency LDHA These patients cannot maintain moderate levels of exercise due to an inability to utilize glycolysis to produce ATP needed for muscle contraction under anaerobic conditions The lactate dehydrogenase reaction is shown in figure 12 where it can be seen that pyruvate reduction to lactate is coupled to the oxidation of NADH to produce NAD When lactate dehydrogenase is not fully functional NADH ogtltidation does not occur at a high enough rate to sustain glycolysis This causes the muscle cells to quickly run out of ATP leading to fatigue and even muscle damage if anaerobic conditions persist Figure 12 Lactate dehydrogenase CH3 CH3 Pyruvate Lactate 8 of 9 pages Bioc 460 Dr Miesfeld Spring 2008 ANSWERS TO KEY CONCEPT QUESTIONS Substrate availabilitv and enzvme activitv levels control olvcolvtic ux bv reoulatino reaction rates Three ofthe ten glycolytic reactions are essentially irreversible under cellular conditions AGltltO and are catalyzed by enzymes that are subject to regulation whereas the other seven reactions are reversible AGO and respond to substrate availability Phosphofructokinase activity is controlled by the negative allosteric effector molecules ATP and citrate which signal high energy charge in the cell and by the positive allosteric effector molecules ADP and F26BP which signal low energy charge Since phosphofructokinase is a key ratelimiting enzyme in the glycolytic pathway the activity level of phosphofructokinase has a major affect on glycolytic flux Glucokinase is subject to both types of control in that it has a low af nity for glucose and is only active when glucose levels are high moreover glucokinase enzyme levels are stimulated by glucose signaling in pancreatic B cells Cvtosolic NAD is required to maintain olvcovtic flux throuoh the olvceraldehvde3P dehydrogenase reaction Glycolysis is the primary source of ATP when oxygen is limiting such as during intense strenuous exercise and also in erythrocytes which lack mitochondria The cytosolic enzyme lactate dehydrogenase regenerates NAD by oxidizing NADH and is highly active under anaerobic conditions due to the buildup of pyruvate which cannot be metabolized to acetyl CoA Individuals that have defects in muscle lactate dehydrogenase cannot sustain strenuous exercise because NAD is not regenerated quickly enough to maintain glycolytic flux and supply the necessary ATP for muscle contraction Yeast alcohol dehydrogenase performs the same function of regenerating NAD to maintain glycolytic ux during the process ofalcoholic fermentation 9 of 9 pages
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