Lecture 23 Quiz next Mon. on Pentose Phosphate Pathway Metabolic regulation and control of glycolysis/gluconeogenesis

Hydrolytic reactions bypass PFK and Hexokinase Instead of generating ATP by reversing the glycolytic reactions, FBP and G6P are hydrolyzed to release Pi in an exergonic reaction. Page 602

Free energies for gluconeogenesis are given in paranthesis. Page 848

2 Pyruvate + 2ATP + 2NADH + 4H+ + 2H2O Glucose + 2ADP + 2Pi + 2NAD+ Glycolysis 2 Pyruvate + 2ATP + 2NADH + 4H+ + 2H2O Glucose + 2ADP + 2Pi + 2NAD+ Gluconeogenesis 2 Pyruvate + 4ATP + 2GTP 2NADH + 4H+ + 6H2O Glucose + 4ADP +2GDP + 6Pi + 2NAD+ Net reaction 2ATP + 2GTP + 4H2O 2ADP + 2GDP + 4Pi

Control Points in Glycolysis

1st reaction of glycolysis (Gº’ = -4 kcal/mol) OH 1 O HO   * 2 3 4 5 6 Glucose Glucokinase (HK IV) in liver Hexokinase (HK) I, II, II Muscle(II), Brain (I) ATP Mg2+ ADP Mg2+ -2O3P-O 6  5 O 4 OH 1 OH * 2 HO  3 Glucose-6-phosphate (G6P) OH

Regulation 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.

Types of regulation Availability of substrate Glucokinase (KM 12 mM) vs. HK (KM = 0.01 - 0.03 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

2 places where there is no net reaction PFK ATP + F-6-P F-1,6-P2 + ADP Mg2+ F-phosphatase 2. F-1,6-P2 F-6-P + Pi Mg2+ Net: ATP ADP + Pi + heat Similar 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.

Enzyme + - Hexokinase G-6-P PFK Pi, ADP, AMP, F-6-P, F-2,6-P2 Primary regulation - reciprocal with energy charge Enzyme + - Hexokinase G-6-P PFK Pi, ADP, AMP, F-6-P, F-2,6-P2 ATP, citrate, NADH F-6-phosphatase ATP AMP, F-2,6-P2 Pyruvate kinase K+, AMP, F-2,6-P2 ATP, acetyl-CoA, cAMP Pyruvate carboxylase Acetyl-CoA

Major regulation is through energy charge ATP Gluconeogenesis Glycolysis ATP ADP Same reactions make AMP or ADP (primarily in lipid and nucleotide metabolism) Adenylate kinase AMP + ATP 2 ADP [ATP] +1/2[ADP] Energy charge [AMP] + [ADP] + [ATP] 1.0 = 100% ATP Body generally likes it close to 0.9 0.5 = 100% ADP 0 = 100% AMP

Control Points in Glycolysis

Enzyme + - Hexokinase G-6-P PFK Pi, ADP, AMP, F-6-P, F-2,6-P2 Primary regulation - reciprocal with energy charge Enzyme + - Hexokinase G-6-P PFK Pi, ADP, AMP, F-6-P, F-2,6-P2 ATP, citrate, NADH F-6-phosphatase ATP AMP, F-2,6-P2 Pyruvate kinase K+, AMP, F-2,6-P2 ATP, acetyl-CoA, cAMP Pyruvate carboxylase Acetyl-CoA

Regulation of PhosphoFructokinase (PFK-1) PKF-1 has quaternary structure Inhibited by ATP and Citrate Activated by AMP and Fructose-2,6-bisphosphate Regulation related to energy status of cell.

PFK-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 ATP AMP and ADP are allosteric activators of PFK. AMP relieves inhibition by ATP. ADP decreases Km for ATP Glucagon (a pancreatic hormone) produced in response to low blood glucose triggers cAMP signaling pathway that ultimately results in decreased glycolysis.

Effect of ATP on PFK-1 Activity Blue no inhiboroty affect. , green inhiboroty.

Effect of ADP and AMP on PFK-1 Activity

Regulation of PFK by Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotes Formed from fructose-6-phosphate by PFK-2 Degraded to fructose-6-phosphate by fructose 2,6-bisphosphatase. In mammals the 2 activities are on the same enzyme PFK-2 inhibited by Pi and stimulated by citrate

Fructose-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+ Glucose PFK-2 F-2,6-P2 F-6-P Bifunctional enzyme ATP - F-6-P + AMP + F-2,6-P2 + Citrate- PEP - PFK-1 F-2,6-Pase cAMP + NTP + F-1,6-Pase F-1,6-P2 AMP- F-2,6-P2-

Glucagon Regulation of PFK-1 in Liver PFK-1 normally inhibited by ATP G-Protein mediated cAMP signaling pathway Induces protein kinase A that activates phosphatase activity and inhibits kinase activity Results in lower F-2,6-P levels decrease PFK-1 activity (less glycolysis)

PFK-2 Serves 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-Pase This 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).

Regulation of Pyruvate Kinase Allosteric enzyme Activated by Fructose-1,6-bisphosphate (example of feed-forward regulation) Inhibited by ATP When 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.

Pyruvate Kinase Regulation

Deregulation 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 supply Tumor cells have fewer mitochondrial, depend more on glycolysis for ATP Increase levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved)

Glycogen biosynthesis Most important storage form of sugar Glycogen - 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.

Figure 18-1a Structure of glycogen. (a) Molecular formula. Page 627

Figure 18-1b. Structure of glycogen Figure 18-1b Structure of glycogen. (b) Schematic diagram illustrating its branched structure. Page 627

Glycogen is an efficient storage form UDP-glucose G-6-P G-1-P + UTP Glycogen + UDP + Pi UDP + ATP UTP + ADP Net: 1 ATP required 90% 1,4 residues Glycogen + Pi G-1-P G-6-P 10% 1,6 residues Glycogen glucose 1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97% efficient for storage of glucose

Glycogen biosynthesis 3 enzymes catalyze the steps involved in glycogen synthesis: UDP-glucose pyrophosphorylase Glycogen synthase Glycogen branching enzyme

Glycogen biosynthesis MgATP HK MgADP Glucose G-6-P [G-1,6-P2] G-1-P phosphoglucomutase F-6-P PGI The hydrolysis of pyrophosphate to inorganic phosphate is highly exergonic and is catalyzed by inorganic pyrophosphatase PPase 2Pi UTP G-1-P PPi UDP-Glucose Pyrophosphorylase

Figure 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

UDP-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.

Glycogen synthase In 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 kinase UDP + ATP UTP + ADP

Figure 18-7 Reaction catalyzed by glycogen synthase. Page 633

Glycogen synthase All carbohydrate biosynthesis occurs via UDP-sugars Can 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.

Glycogen branching Catalyzed by amylo (1,41,6)-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.

Figure 18-8 The branching of glycogen. Page 634

Glycogen Breakdown Requires 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).

Glycogen 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 regulation Phosphorylase a- has a phosphoryl group on Ser14 in each subunit. Phosphorylase b-lacks the phosphoryl groups. Inhibitors: ATP, G6P, glucose Activator: AMP Glycogen 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

Figure 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

Page 630

Phosphoglucomutase Converts G1P to G6P. Reaction is similar to that of phosphoglycerate mutase Difference 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.

Figure 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

Glycogen debranching enzyme (14) transglycosylase (glycosyl transferase) transfers a (14) linked trisaccharide unit from a limit branch to a nonreducing end of another branch. Forms a new (14) linkage with three more units available for phosphorylase. The (16) bond linking the remaining linkage is hydrolyzed by the same enzyme to yield glucose. 2 active sites on the same enzyme.

Figure 18-5 Reactions catalyzed by debranching enzyme. Page 631

Requires G6P for activation Regulation of glycogen synthesis Both synthase & phosphorylase exist in two forms. Phosphorylated at Ser residues by synthase kinase and phosphorylase kinase Synthase a Normal form “active” Synthase b Requires G6P for activation “inactive” OH OP Pi ATP phosphoprotein phosphatase Synthase kinase ADP

Independent of energy status Regulation of glycogen synthesis AMP+, ATP-, G6P- Phosphorylase b Normal form “inactive” OH Pi OH ATP phosphorylase kinase phosphoprotein phosphatase Ca2+ ADP Phosphorylase a Independent of energy status active OP OP High [ATP] (related to high G6P) inhibits phosphorylase and stimulates glycogen synthase.

Regulation of glycogen synthesis Process is also under hormonal control Adrenaline (epinephrine) can regulate glycogen synthesis/breakdown by stimulating adenylate cyclase ATP 1. External stimulus Adrenaline Adenylate cyclase cAMP 2. R2C2 cAMP + PPi cAMP dependent protein kinase [C]2 + [R-AMP]2 “inactive” “active” ATP ADP 3a. Glycogen synthase a (active) Glycogen synthase b (inactive) [C]2 ATP ADP 3b. Inactive phosphorylase kinase Active phosphorylase kinase [C]2 ATP ADP Phosphorylase b (inactive) Phosphorylase a (active)

Consider the whole system Resting muscle O2 Glycolytic pathway pyruvate respiration ATP Inactive phosphorylase b, active synthase a Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present Upon stress Epinephrine cAMP Synthase/phosphorylase kinase Phosphorylse b Phosphorylse a

Figure 23-25 The pentose phosphate pathway. Page 863