61st reaction of glycolysis (Gº’ = -4 kcal/mol) OH1OHO*23456GlucoseGlucokinase (HK IV) in liverHexokinase (HK) I, II, IIMuscle(II), Brain (I)ATPMg2+ADPMg2+-2O3P-O65O4OH1OH*2HO3Glucose-6-phosphate (G6P)OH
7Regulation of Hexokinase Glucose-6-phosphate is an allosteric inhibitor of hexokinase.Levels of glucose-6-phosphate increase when downstream steps are inhibited.This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis.Hexokinase is not necessarily the first regulatory step inhibited.
8Types of regulationAvailability of substrate Glucokinase (KM 12 mM) vs. HK (KM = mM)Compartmentalization -Brain vs. Liver vs. Muscle (type I mitochondrial membrane, type II cytoplasmic)Allosteric regulation - feedback inhibition by G-6-P, overcome by Pi in type I (Brain/ mitochondrial controlled by Pi levels)Hormonal regulation. Liver has HK as fetal tissue. Changes to glucokinase after about 2 weeks. If there is no dietary carbohydrate, no glucokinase. Must have both insulin and carbohydrates to induce.Much lower substrate concentration to reach vmax
92 places where there is no net reaction PFKATP + F-6-PF-1,6-P2 + ADPMg2+F-phosphatase2. F-1,6-P2F-6-P + PiMg2+Net: ATPADP + Pi + heatSimilar reaction occurs with hexokinase and G-6-phosphatase.Generally regulated so this does not occur (futile cycle).May function in hibernating animals to generate heat.
11Major regulation is through energy charge ATPGluconeogenesisGlycolysisATPADPSame reactions make AMP or ADP (primarily in lipid and nucleotide metabolism)Adenylate kinaseAMP + ATP2 ADP[ATP] +1/2[ADP]Energy charge[AMP] + [ADP] + [ATP]1.0 = 100% ATP Body generally likes it close to 0.90.5 = 100% ADP0 = 100% AMP
14Regulation of PhosphoFructokinase (PFK-1) PKF-1 has quaternary structureInhibited by ATP and CitrateActivated by AMP and Fructose-2,6-bisphosphateRegulation related to energy status of cell.
15PFK-1 regulation by adenosine nucleotides ATP is substrate and inhibitor. Binds to active site and allosteric site on PFK. Binding of ATP to allosteric site increase Km for ATPAMP and ADP are allosteric activators of PFK.AMP relieves inhibition by ATP.ADP decreases Km for ATPGlucagon (a pancreatic hormone) produced in response to low blood glucose triggers cAMP signaling pathway that ultimately results in decreased glycolysis.
16Effect of ATP on PFK-1 Activity Blue no inhiboroty affect. , green inhiboroty.
18Regulation of PFK by Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotesFormed from fructose-6-phosphate by PFK-2Degraded to fructose-6-phosphate by fructose 2,6-bisphosphatase.In mammals the 2 activities are on the same enzymePFK-2 inhibited by Pi and stimulated by citrate
19Fructose-2,6-bisphosphate can override Energy charge Produced when [glucose] is high but need glycolysis for anabolic role.When glucose is needed by the brain (about 120 g/day via diet or other tissues)3 PGA -cAMP -Citrate+GlucosePFK-2F-2,6-P2F-6-PBifunctional enzymeATP -F-6-P +AMP +F-2,6-P2 +Citrate-PEP -PFK-1F-2,6-PasecAMP +NTP +F-1,6-PaseF-1,6-P2AMP-F-2,6-P2-
20Glucagon Regulation of PFK-1 in Liver PFK-1 normally inhibited by ATPG-Protein mediated cAMP signaling pathwayInduces protein kinase A that activates phosphatase activity and inhibits kinase activityResults in lower F-2,6-P levels decrease PFK-1 activity (less glycolysis)
21PFK-2Serves to override ATP inhibition and promote glycolysis once intermediates build up [citrate] [PEP][GAP]Block PFK-2 activity with high [NTP] by stimulating F-2,6-PaseThis will break down F-2,6-P2 and restores energy charge regulation.cAMP is the hormonal control. The presence of cAMP is indicative of low blood sugar (glucagon) stimulates F-2,6-Pase to increase F-6-P formtion for gluconeogenesis (cAMP also inhibits Pyruvate Kinase).
22Regulation of Pyruvate Kinase Allosteric enzymeActivated by Fructose-1,6-bisphosphate (example of feed-forward regulation)Inhibited by ATPWhen high fructose 1,6-bisphosphate present plot of [S] vs Vo goes from sigmoidal to hyperbolic.Increasing ATP concentration increases Km for PEP.In liver, PK also regulated by glucagon. Protein kinase A phosphorylates PK and decreases PK acitivty.
24Deregulation of Glycolysis in Cancer Cells Glucose uptake and glycolysis is 10X faster in solid tumors than in non-cancerous tissues.Tumor cells initally lack connection to blood supply so limited oxygen supplyTumor cells have fewer mitochondrial, depend more on glycolysis for ATPIncrease levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved)
25Glycogen biosynthesis Most important storage form of sugarGlycogen - highly branched (1 per 10) polymer of glucose with (1,4) backbone and (1,6) branch points. More branched than starch so more free ends.Average molecular weight -several million in liver, muscle.1/3 in liver (more concentrated but less overall mass (5-8%)),2/3 in muscle (1%).Not found in brain - brain requires free glucose (120 g/ day) supplied in diet or from breakdown of glycogen in the liver.Glucose levels regulated by several key hormones - insulin, glucagon.
26Figure 18-1a Structure of glycogen. (a) Molecular formula. Page 627
27Figure 18-1b. Structure of glycogen Figure 18-1b Structure of glycogen. (b) Schematic diagram illustrating its branched structure.Page 627
28Glycogen is an efficient storage form UDP-glucoseG-6-PG-1-P + UTPGlycogen + UDP + PiUDP + ATPUTP + ADPNet: 1 ATP required90% 1,4 residuesGlycogen + PiG-1-PG-6-P10% 1,6 residuesGlycogenglucose1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97% efficient for storage of glucose
29Glycogen biosynthesis 3 enzymes catalyze the steps involved in glycogen synthesis:UDP-glucose pyrophosphorylase Glycogen synthaseGlycogen branching enzyme
30Glycogen biosynthesis MgATPHKMgADPGlucoseG-6-P[G-1,6-P2]G-1-PphosphoglucomutaseF-6-PPGIThe hydrolysis of pyrophosphate to inorganic phosphate is highly exergonic and is catalyzed by inorganic pyrophosphatasePPase2PiUTPG-1-PPPiUDP-Glucose Pyrophosphorylase
31Figure 18-6 Reaction catalyzed by UDP–glucose pyrophosphorylase. The reaction is a phosphoanhydride exchange in which the phosphoryl oxygen of G1P attacks the alpha phosphorous atom of UTP to form UDPG and release Ppi. This is then hydrolyzed by inorganic pyrophosphatse.Page 633
32UDP-Glucose pyrophosphorylase Coupling the highly exergonic cleavage of a nucleoside triphosphate to form PPi is a common biosynthetic strategy.The free energy of the hydrolysis of PPi with the NTP hydrolysis drives the reaction forward.
33Glycogen synthaseIn this step, the glucosyl unit of UDP-glucose (UDPG) is transferred to the C4-OH group of one of glycogen’s nonreducing ends to form an (1,4) glycosidic bond.Involves an oxonium ion intermediate (half-chair intermediate)Each molecule of G1P added to glycogen regenerated needs one molecule of UTP hydrolyzed to UDP and Pi.UTP is replenished by nucleoside diphosphate kinaseUDP + ATPUTP + ADP
34Figure 18-7 Reaction catalyzed by glycogen synthase. Page 633
35Glycogen synthaseAll carbohydrate biosynthesis occurs via UDP-sugarsCan only extend an already (1,4) linked glucan change.First step is mediated by glycogenin, where glucose is attached to Tyr 194OH group.The protein dissociates after glycogen reaches a minimum size.
36Glycogen branchingCatalyzed by amylo (1,41,6)-transglycosylase (branching enzyme)Branches are created by the terminal chain segments consisting of 7 glycosyl residues to the C6-OH groups of glucose residues on another chain.Each transferred segment must be at least 11 residues.Each new branch point at least 4 residues away from other branch points.
38Glycogen BreakdownRequires 3 enzymes:Glycogen phosphorylase (phosphorylase) catalyzes glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1-phosphate (G1P)Glycogen debranching enzyme removes glycogen’s branches, allowing glycogen phosphorylase to complete it’s reactions. It also hydrolyzes a(16)-linked glucosyl units to yield glucose. 92% of glycogen’s glucse residues are converted to G1P and 8% to glucose.Phosphoglucomutase converts G1P to G6P-can either go through glycolysis (muscle cells) or converted to glucose (liver).
39Glycogen Phosphorylase A dimer - 2 identical 842 residue subunits.Catalyzes the controlling step of glycogen breakdown.Regulated by allosteric interactions and covalent modification.Two forms of phosphorylase made by regulationPhosphorylase a- has a phosphoryl group on Ser14 in each subunit.Phosphorylase b-lacks the phosphoryl groups.Inhibitors: ATP, G6P, glucoseActivator: AMPGlycogen forms a left-handed helix with 6.5 glucose residues per turn.Structure can accommodate 4-5 sugar residues only.Pyridoxal phosphate is an essential cofactor for phosphorylase.Converts glucosyl units of glycogen to G1P
40Figure 18-2a. X-Ray structure of rabbit muscle glycogen phosphorylase Figure 18-2a X-Ray structure of rabbit muscle glycogen phosphorylase. (a) Ribbon diagram of a phosphorylase b subunit.Page 628
42PhosphoglucomutaseConverts G1P to G6P.Reaction is similar to that of phosphoglycerate mutaseDifference between phosphoglycerate mutase and phosphoglucomutase is the amino acid residue to which the phosphoryl group is attached.Serine in phosphoglucomutase as opposed to His imidazole N in phosphoglycerate mutase.G1,6P occasionally dissociates from the enzyme, so catalytic amounts are necessary for activity. This is supplied by the enzyme phosphoglucokinase.
43Figure 18-4 The mechanism of action of phosphoglucomutase. The OH group at C1 of G6P attacks the phosphoenzyme to form a dephosphoenzyme g1,6 intermediate.Ther Ser OH group on the dephosphoenzyme attacks the phosphoryl group at C6 to regenerate the phosphoenzyme and G1P.Page 631
44Glycogen debranching enzyme (14) transglycosylase (glycosyl transferase) transfers a (14) linked trisaccharide unit from a limit branch to a nonreducing end of another branch.Forms a new (14) linkage with three more units available for phosphorylase.The (16) bond linking the remaining linkage is hydrolyzed by the same enzyme to yield glucose.2 active sites on the same enzyme.
45Figure 18-5 Reactions catalyzed by debranching enzyme. Page 631
46Requires G6P for activation Regulation of glycogen synthesisBoth synthase & phosphorylase exist in two forms. Phosphorylated at Ser residues by synthase kinase and phosphorylase kinaseSynthase aNormal form“active”Synthase bRequires G6P for activation“inactive”OHOPPiATPphosphoproteinphosphataseSynthase kinaseADP
47Independent of energy status Regulation of glycogen synthesisAMP+, ATP-, G6P-Phosphorylase bNormal form“inactive”OHPiOHATPphosphorylase kinasephosphoproteinphosphataseCa2+ADPPhosphorylase aIndependent of energy statusactiveOPOPHigh [ATP] (related to high G6P) inhibits phosphorylase and stimulates glycogen synthase.
48Regulation of glycogen synthesis Process is also under hormonal controlAdrenaline (epinephrine) can regulate glycogen synthesis/breakdown by stimulating adenylate cyclaseATP1. External stimulusAdrenalineAdenylate cyclasecAMP2. R2C2cAMP + PPicAMP dependent protein kinase[C]2 + [R-AMP]2“inactive”“active”ATPADP3a. Glycogen synthase a(active)Glycogen synthase b (inactive)[C]2ATPADP3b. Inactive phosphorylase kinaseActive phosphorylase kinase[C]2ATPADPPhosphorylase b(inactive)Phosphorylase a (active)
49Consider the whole system Resting muscle O2 Glycolytic pathway pyruvaterespirationATPInactive phosphorylase b, active synthase aMuscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 presentUpon stressEpinephrinecAMPSynthase/phosphorylase kinasePhosphorylse bPhosphorylse a
50Figure 23-25 The pentose phosphate pathway. Page 863