Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler GLYCOLYSIS AND GLUCONEOGENESISl Related Reading Chapter 16 434452 in Stryer 6m edition OBJECTIVES 1 For reactions in the glycolysis pathway a describe the reactions for those that catalyze steps in which ATP is used b describe the reactions for those that catalyze steps that produce ATP for cell energy c identify the reaction that requires NAD produces NADH and the high energy phosphate produced by this reaction 2 Explain why NAD must be regenerated for glycolysis and how that is accomplished under anaerobic conditions 3 Discuss the metabolic role of niacin 4 Differentiate between the characteristics of the H4 LDHI and M4 LDH5 isozymes of lactate dehydrogenase PHYSIOLOGICAL PREMISE What biochemical properties in the liver allow us to handle a large dietary intake of glucose without becoming severely hyperglycemic First to ensure that glucose can readily enter the liver its transport is independent of any metabolic controls Second as soon as glucose enters the liver it is quickly phosphorylated by a kinase enzyme that has a high capacity for handling the glucose Why is immediate phosphorylation of glucose important Recall that phosphate contains several negative charges Since membranes are hydrophobic they do not readily permit penetration of molecules bearing charges unless there is a speci c transport protein to permit their movement across the membrane Hence it is these conditions in the liver that contribute to the rapid removal of glucose as it passes the liver immediately after entering the circulation from intestinal cells THE GLYCOLYTIC PATHWAY you will NOTbe tested on chemical structures The Overall Pathway The pathway contains three phases The rst phase traps the glucose by phosphorylation and then primes the molecule for splitting by addition of a second phosphate This phase costs the cell 2 ATP per glucose molecule The second phase involves splitting the sixcarbon molecule into two phosphorylated triose molecules The third phase includes an oxidation step and ATP formation with the nal product being pyruvate Under anaerobic conditions pyruvate is converted to lactate while under aerobic conditions the pyruvate is oxidized to C02 via pyruvate dehydrogenase and the citric acid cycle However in some tissues such as liver and adipose tissue pyruvate may not be oxidized completely since two of its three carbons may be used for the synthesis of fatty acids lipogenesis All intermediates of glycolysis carry phosphate groups to lock them in the cell Phase 1 ofGlycalysis Hexokinaseglucokinase see Fig 1A CHZOH CHZOP03239 Glucose ATP Glucose 6phosphate ADP H gt H OH OH OH OHOH OH Glycolysisgluconeogenesis ll Glucose and other hexoses are phosphorylated at the expense of ATP with a requirement for MgH Thus the glycolytic pathway begins with energy expenditure but it is a worthwhile investment Hexokinase catalyzes the irreversible phosphorylation of hexoses in general and is found in most cells the exceptions being liver and pancreas Hexokinase has a low Km making it active even at low concentrations of glucose In contrast glucokinase is glucose speci c and is found primarily in liver and pancreas In contrast to hexokinase it has a high Km to ensure a proportionate response in the liver to elevation of portal glucose from dietary sources and in the pancreas to foster signaling of the need for insulin Both reactions are unidirectional irreversible thus trapping glucose in the cell in its phosphorylated form Note that kimise reactions always useAT P as the source ofphosphate When inorganic phosphate is used the reaction is catalyzed by aphasphmyluse The glucose6 phosphate product is isomerized to fructose6phosphate creating a hexose containing a keto group from one containing an aldehyde Phosphofructokinase l see Fig 1A CHZOPOsz39 CHZOPOsz39 2 O H20H gt O Hzopos Fructose 6phosphate ATP gt H H Fructose 16bisphosphate ADP OH OH OH OH PFKl is so designated to indicate that it irreversibly attaches the terminal phosphate from ATP to the C1 position of fructose6phosphate Later we will discuss the PFK2 enzyme PFKl involves expenditure of a second molecule of ATP and because of a large free energy difference this reaction is physiologically irreversible It is the rate limiting enzyme ofglycolysis and the major regulated step Phase 2 ofGlycalysis Aldolase A Fructose 16bisphosphate Dihydroxyacetonephosphate Glyceraldehyde3phosphate 2 CH2P03 HZOPOSZ HZOPng39 o H H 0 H OH OH OH CHZOH CHZOPO3239 This reversible reaction completes the rst half of glycolysis hexose to two triose phosphates Fructose 16 bisphosphate is split into two trioses Note that the phosphorylated carbon that is boldfaced appears in dihydroxyacetonephsophate and the other phosphorylated italicized carbon appears in glyceraldehyde3 phosphate These trioses are interconverted by triose phosphate isomerase to produce a single product glyceraldehyde3P Thus there are two molecules of glyceraldehyde3P formed to continue through glycolysis This reaction is reversible and operates in the other direction during gluconeogenesis Phase 3 ofGlycalysis leceraldehvde 3 phosphate dehvdrogenase see Fig 1B Glyceraldehyde3phosphate NAD Pi 13Bisphosphoglycerate NADH o H 0 0103 H OH 4 H OH HzOPng39 HZOPng39 Glycolysisgluconeogenesis l2 This reversible step is an oxidationreduction reaction with production of NADH The hydrogen boldfaced added to form NADH is derived from the aldehyde group of the glyceraldehyde3phosphate The NADH formed must be reoxidized to regenerate NAD for sustaining glycolysis Under anaerobic conditions pyruvate is reduced to lactate Fig 1B Under aerobic conditions mitochondrial systems oxidize NADH and produce ATP Energy released from the reaction is conserved as a high energy phosphate bond in 13 bisphosphoglycerate Inorganic phosphate Pi rather than ATP provides the source of the phosphoryl group that is attached to carbon 1 This reaction also can operate in reverse during gluconeogenesis as do all of the remaining reactions except for pyruvate kinase the last step in glycolysis Niacin is the functional component of certain coenzymes ie NADT NADH NADPT NADPH required for dehydrogenase reactions such as glyceraldehyde3phosphate dehydrogenase and lactate dehydrogenase discussed below We will encounter other dehydrogenases involved in energy metabolism of carbohydrates and fatty acids Under aerobic conditions NADH is oxidized via the respiratory electron transport chain to produce large amounts of ATP Niacin is also important in pigment metabolism and biosynthesis of lipids and glucose Niacin s functions account for the consequences of its de ciency Niacin de ciency is pellagra which is characterized by the quot4 Dsquot diarrhea dermatitis dementia and death Symptoms include muscular weakness anorexia indigestion and skin eruptions all linked to insuf cient production of energy lipids and pigments Lesions in the central nervous system lead to confusion disorientation and neuritis Digestive abnormalities cause irritation and in ammation of the mucous membranes of the mouth and gastrointestinal tract Generally niacin is in an unavailable form in corn however the treatment of corn tortillas with lime water alkali releases the niacin Similarly roasting coffee beans releases niacin from quottrigonellinquot a bound form of niacin Phosphoglycerate kinase see Fig 1B 0 0103 0 OH H OH H OH 13bisphosphoglycerate ADP lt 3 hos ho l cerateATP HZOPOf39 HZOPOf p p gy 13Bisphosphoglycerate a high energy phosphorylated intermediate drives the phosphorylation of ADP to ATP The high energy phosphate bond attached to carbonl is cleaved and the phosphate transferred to ADP to form ATP Recall that two triose molecules are proceeding through glycolysis so that this step recovers the 2 ATP used in the rst half of the glycolytic pathway This is an example of substrate level phosphorylation The reactions that immediately follow phosphoglycerate kinase a mutase and an enolase form 2 phosphoglycerate and phosphoenolpyruvate respectively Phosphoenolpyruvate a very high energy phosphate compound The bond energy in phosphoenolpyruvate is double that in the terminal bond of ATP Pyruvate kinase see Fig 1B O OH O OH 2 gt Phosphoenolpyruvate ADP Pyruvate ATP 1303 0 H2 H3 This reaction is another example of substrate level phosphorylation Since two molecules of phosphoenolpyruvate are derived from the original glucose a net 2 ATP are produced The reaction is physiologically irreversible and is highly regulated The pyruvate product may be oxidized in the mitochondria aerobic glycolysis or reduced to lactate via lactate dehydrogenase anaerobic glycolysis Glycolysisgluconeogenesis l3 Figure 1A The rst two phases of the glycolytic pathway Though several of these enzymes are reversible all the reactions are drawn solely as they occur during glycolysis The teXt describes which are irreversible and which are reversible Glucose ATP Glucokinase is used for the rst reaction in liver m parenchyma cells and pancreatic betacells ADP Glucose6phosphate gt Fructose6phosphate Phosphoglucose lsomerase Phosphofructokinase l ADP The phosphorylamd Fructose16b1sphosphate fructose is split Aldolasel Glyceraldehyde 3 P Dihydroxyacetone phosphate Glyceraldehyde3 phosphate D1hydroxyacetone phosphate Trio provides one molecule and the Phosphate other is a product of Aldolase A zsomerase Glyceraldehyde3phosphate 392 molecules of Glyceraldehyde3phosphate 1 Second half of glycolysis Glycolysisgluconeogenesis l4 Figure 1B The third reductionoxidation and ATP formation phase of the glycolytic pathway showing the aerobic and anaerobic fates of pyruvate Recall that 2 molecules of glyceraldehyde enter from the rst half of glycolysis so that every reaction here must be doubled Except for pyruvate kinase all other reactions are reversible but here all the reactions are drawn solely as they occur during glycolysis Recall that two molecules enter the Glyceraldehyde3phOSphate second half of glycolysis so that from rst half of glycolysis everything is doubled G3PDH Redox step of glycolysis for which NAD must be regenerated i NAD Pi Glyceraldehyde 3 phosphate dehydrogenase NADH PF PG Kinase First site of ATP formation by substratelevel phosphorylation 7 2ATP used in the rst half of glycolysis are replaced here 13Bisphosphoglycerate 7 3Phosphoglycerate Phospho P glycerate glycerol ADP kinase ATP mutase E nolase Phosphoenolpyruvate4 2Phosphoglycerate ADP PK Second site of ATP Pyruvate kinase formation yielding a net gain of 2 ATP ATP NAD NADH PF Lactate 5 4 Pyruvate gt C02 Lactate Pyruvate dehydrogenase dehydrogenase citric acid cycle NAD must be regenerated for the glyceraldehyde3P dehydrogenase reaction either via lactate dehydrogenase anaerobic metabolism or via aerobic metabolism to C02 Glycolysisgluconeogenesis l5 Lactate Dehydragenase see Fig 1B FF OH O 39OH pyruvate NADH HT lt gt lactate NADT 310 HEDH anaerobic glycolysis H3 H3 Lactate dehydrogenase under anaerobic conditions reduces pyruvate to lactate The two hydrogen atoms added to pyruvate to form lactate are derived from NADH and a proton H This step regenerates NAD for the glyceraldehyde3phosphate dehydrogenase reaction otherwise glycolysis would stop The enzyme is a tetramer consisting of H andor M subunits There are ve possible combinations including H4 LDHI H3M1 LDHZ HzMz LDHs H1M3 LDH4 M4 LDH5 The M4 isozyme is the principal form found in anaerobic skeletal muscles such as those used in sprinting and in red blood cells because they lack mitochondria The M4 isozyme preferentially produces lactate and is therefore the key form linked to anaerobic glycolysis and the source of regenerating the NADT to sustain the glyceraldehyde3phosphate dehydrogenase reaction It functions especially well when the concentration of pyruvate is high in the cytoplasm as occurs during anaerobic glycolysis This ensures that lactate dehydrogenase regenerates the NAD needed for glycolysis H4 and H3M1 are the principal isozyme forms found in heart and aerobic skeletal muscles such as those used in a marathon run Compared to the M4 isozyme the H4 form has a higher affinity for lactate lower Km and is allosterically inhibited by pyruvate Consequently during glycolysis it favors the oxidation of lactate to pyruvate and prevents the conversion of pyruvate to lactate This eXplains why it is found in very aerobic tissues that use mitochondrial metabolism to regenerate NAD for glycolysis rather than lactate dehydroegnase FRUCTOSE METABOLISM IN LIVER Fructokinase see Fig 2 CHZOH CHZOH O HZOH O HZOPOf39 Fructose ATP Fructose lphosphate ADP H H OH OH OH OH This is the predominant pathway in liver for the metabolism of fructose from the diet Fructokinase catalyzes the phosphorylation offructose on the C1 one position Do not confuse this enzyme with phosphofructokinase Other tissues eg muscle brain utilize hexokinase to phosphorylate fructose on the C6 position In those tissues fructose 6phosphate enters directly into glycolysis However the vast majority of fructose is processed in the liver with very minimal amounts in all other tissues Aldolase B see Fig 2 CHZOH 2 H OPO 2 Fructose lphosphate o Hzopos 39 gt 2 3 O H Glyceraldehyde H O H OH Dihydroxyacetone phosphate OH OH CHZOH HZOH Glycolysisgluconeogenesis l6 For all of the carbons from fructose to proceed through the rest of glycolysis both products of this reaction must be converted to glyceraldehyde 3phosphate Dihydroxyacetone phosphate is isomerized to glyceraldehyde3 phosphate Glyceraldehyde is phosphorylated by a speci c kinase to form glyceraldehyde3phosphate as well Hence fructose enters liver glycolysis as 2 molecules of glyceraldehyde3phosphate that can then proceed through the second half of glycolysis Fig 1B The fructose pathway consumes 2 ATP just as does the rst half of glycolysis The 4 ATP produced in the second half of glycolysis then yield a net 2 ATP from fructose metabolism to pyruvate Fructokinase FRUCTOSE ATP gt FRUCTOSE1P ADP A AldolaseB Fructose1phosphate aldolase GLYCERALDEHYDE DIHYDROXYACETONE PHOSPHATE Kinase ATP lsomerase GLYCOLYSS 2 GLYCERALDEHYDESPHOSPHATE Figure 2 Principal liver pathway for fructose metabolism The endproducts of fructose metabolism are converted to two molecules of glyceraldehyde3phosphate that can then enter glycolysis GALACTOSE METABOLISM IN LIVER Galactose Pathway Galactokinase see Fig 3 Galactose ATP gt Galactose lphosphate ADP As in the case of fructose this speci c kinase is found in liver and yields the C1 phosphorylated product Uridyl transferase see Fig 3 UDPglucose is an activated form of glucose found as an intermediate in glycogen formation UDPglucose is recycled from UDPgalactose via an epimerase Thus there is no net change in the concentration of this compound The product glucose lP is an intermediate in glycogen formation and breakdown and is readily converted by phosphoglucomutase into glucose 6phosphate for entry into glycolysis Thus galactose enters glycolysis early in the rst half bypassing only the hexokinaseglucokinase step The galactose pathway coupled with glycolysis yields a net two ATP in the conversion of galactose to pyruvate Glycolysisgluconeogenesis l7 Galactose ATP Galactokinase ADP Figure 3 Principal pathway for metabolism of galactose located in the liver The endproduct glucose 6phosphate provides the means of entry into glycolysis Galactose1P UDPGlucose V Uridyl transferase Phosphoglucomutase UDPGalactose 1 GIucose6P Eplmerase ix 39 UDPGlucose GLYCOLYSIS Galactasemia A defect of the u1idyltyransferase reaction produces a serious condition galactosemia elevated blood galactose Lack of uIidyltransferase causes both galactoselphosphate and galactose to accumulate Accumulation of galactoselphosphate in the liver causes trapping of phosphate that becomes unavailable for ATP formation thereby leading to energy depletion in the liver In the lens of the eye failure to process galactose due to the transferase defect leads to formation of galactitol galactose NADPH gt galactitol NADP Galactitol accumulation leads to diffusion of water into the lens of the eye initiating formation of cataracts even in infants Glycolysisgluconeogenesis l8
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