Storage Mechanisms and Control in Carbohydrate Metabolism

I. How Glycogen Is Produced and Degraded

■ A supply of exceed glucose store glucose as a polymer (glycogen).

■ Degradation of glycogen Several glucose residues released simultaneously. Useful to meet short-term demands for energy by

increasing the glucose supply. ■ Optimized for its ability (store; deliver energy quickly) average chain length of the branches (13 residues).

1. Glycogen breakdown

■ Glycogen is found primarily in liver and muscle. ■ Low levels of glucose in blood glycogen breakdown G-6-P glucose. ■ In muscle glycogen breakdown G-6-P glycolytic pathway directly. ■ Three reactions play roles in the conversion of glycogen to G-6-P.

(1) Each glucose residue cleaved from glycogen reacts with phosphate to give G-1-P (phosphorolysis).

(2) G-1-P isomerizes to give G-6-P.

(3) Complete breakdown of glycogen - Requires a debranching reaction at branch points. - Glycogen phosphorylase; phosphoglucomutase. Glycogen phosphorylase: Glycogen + Pi G-1-P + Remainder of

glycogen Phosphoglucomutase: G-1-P G-6-P

■ Glycogen phosphorylase (1) Cleaves the α (14) linkages. (2) Complete breakdown requires debranching enzymes that degrade the

α(16) linkages. (3) No ATP is hydrolyzed in the first reaction saves 1 ATP/glucose a net gain of 3 ATP/glucose

(4) The debranching of glycogen involves the transfer of a "limit branch" of 3 glucose residues to the end of another branch. Subsequently removed by glycogen phosphorylase. The same glycogen debranching enzyme then hydrolyzes the α(16)

glycosidic bond of the last glucose residue remaining at the branch point.

■ When an organism needs energy quickly, glycogen breakdown is important. Muscle tissue can mobilize glycogen more easily than fat and can do so

anaerobically.

2. Glycogen production

■ The synthesis of glycogen requires energy provided by the hydrolysis of a nucleoside triphosphate, UTP.

(1) G-1-P (obtained from G-6-P by an isomerization reaction) reacts with UTP to produce UDPG and pyrophosphate (PPi).

Enzyme: UDPG pyrophosphorylase. Exchange of 1 phosphoric

anhydride bond for another: free-energy change close to zero.

Hydrolysis of pyrophosphate to 2 phosphates: a strongly exergonic reaction.

Supply of UTP is replenished by an exchange reaction with ATP (by nucleoside phosphate kinase): UDP + ATP UTP + ADP

(2) The addition of UDPG to a growing chain of glycogen. - Each step involves formation of a new α (14) glycosidic bond - Catalyzed by the enzyme glycogen synthase.

♣ Glycogen synthase

- Cannot simply form a bond between two isolated glucose molecules. - It must add to an existing chain with α (14) glycosidic linkages. - The initiation of glycogen synthesis requires a primer for this reason. - The -OH group of a specific Tyr of the protein glycogenin (37,300 Da) serves this purpose.

♣ Glycogen synthesis (1) A glucose residue is linked to the -OH group of a specific Tyr of the

protein glycogenin (a glycosyltransferase) and glucose residues are successively added to this first one.

(2) UDPG is the donor in this autoglycosylation. (3) Glycogenin molecule itself acts as the catalyst for addition of glucoses

until there are about 8 of them linked together. (3) At that point, glycogen synthase takes over.

(3) Synthesis of glycogen requires the formation of α(16) as well as α(14) glycosidic linkages.

- A branching enzyme accomplishes this task. - Transferring a segment about 7 residues long from the end of a

growing chain to a branch point where it catalyzes the formation of the required α(16) glycosidic linkage.

- Note that this enzyme has already catalyzed the breaking of an α (14) glycosidic linkage in the process of transferring the oligosaccharide segment.

- Each transferred segment must come from a chain at least 11 residues long; each new branch point must be at least 4 residues away from the nearest existing branch point.

3. Balancing glycogen breakdown and production

■ How does an organism ensure that glycogen synthesis and glycogen breakdown do not operate simultaneously?

■ A major controlling factor lies in the behavior of glycogen phosphorylase. Allosteric control and covalent modification.

(1) In liver, Glucose is an allosteric inhibitor of phosphorylase a transition to the T state shifts the equilibrium to phosphorylase b.

(2) In muscle, - The primary allosteric effectors are ATP, AMP, and G6P. - Use ATP Increase in AMP stimulates formation of the R state of

phosphorylase b. - ATP is plentiful or glucose-6-phosphate builds up act as allosteric

inhibitors back to the T form. (3) The combination of covalent modification and allosteric control of the p

rocess allows for a degree of fine-tuning that would not be possible with either mechanism alone.

■ The activity of glycogen synthase is subject to the same type of covalent modification as glycogen phosphorylase.

(1) The difference is that the response is opposite. (2) Glycogen synthase: Phosphorylated inactive form, unphosphorylated active form.

(3) Hormonal signal (glucagons, epinephrine) stimulate the phosphorylation of glycogen synthase inactive; at the same time hormonal signal activating phosphorylase.

(4) Allosteric control: ATP (inhibitor), G6P (activator). - Phosphorylated (inactive) form [glycogen synthase D] active only under very high concentrations of G6P.

- Nonphosphorylated form [glycogen synthase I] active even with low concentrations of G6P.

II. Gluconeogenesis Produces Glucose from Pyruvate

■ The conversion of pyruvate to glucose.

■ Not the exact reversal of glycolysis.

■ Some of the reactions of glycolysis irreversible bypassed in

gluconeogenesis.

(1) The production of pyruvate (and ATP) from phosphoenolpyruvate..

(2) The production of fructose-1,6-bisphosphate from fructose-6-phosphate.

(3) The production of glucose-6-phosphate from glucose.

1. Pyruvate to phosphoenolpyruvate in two steps

■ Oxaloacetate is an intermediate in gluconeogenesis

A. Pyruvate oxaloacetate(OA)

(1) Pyruvate carboxylase (PC): an allosteric enzyme found in the mitochondria.

- Acetyl-CoA (allosteric effector) activates PC.

(i) High levels of acetyl-CoA: pyruvate (a precursor of acetyl-CoA)

diverted to gluconeogenesis.

(ii) Mg2+ and biotin are also required for effective catalysis.

(2) Biotin is a carrier of CO2

(i) It has a specific site for covalent attachment of CO2.

(ii) Carboxyl group of the biotin amide bond with the E-amino group of a

specific lysine side chain of PC.

(iii) CO2 attached to the biotin shifted to pyruvate to form oxaloacetate.

(iv) Note that ATP is required for this reaction.

B. Oxaloacetate(OA) phosphoenolpyruvate

(1) Phosphoenolpyruvate carboxykinase (PEPCK): found in the mitochondria

and the cytosol.

(2) Involves hydrolysis of GTP.

■ The successive carboxylation and decarboxylation reactions are both close

to equilibrium (low values of their standard free energies)

the conversion of pyruvate to phosphoenolpyruvate is also close to

equilibrium (∆G°́ = 2.1 kJ/mol = 0.5 kcal/mol).

A small increase in the level of OA ↔ a small increase in the level of PEP.

■ OA (formed in the mitochondria) two fates with respect to gluconeogenesis. (1) It can continue to form PEP leave the mitochondria via a specific transporter gluconeogenesis in the cytosol.

(2) OA malate leave the mitochondria OA (by cytosolic malate dehydrogenase). OA cannot leave the mitochondria, but malate

can. (The pathway involving malate is the one that takes place in the liver, where gluconeogenesis largely takes place).

■ Why these two paths exist to get PEP into the

cytosol to continue gluconeogenesis? The purpose of the roundabout way of getting OA

out of the mitochondria via malate dehydrogenase is to produce NADH in the cytosol so that gluconeogenesis can continue.

2. Dephosphorylation of sugar phosphates

■ Other two reactions A phosphate-ester bond to a sugar-hydroxyl group is hydrolyzed. Catalyzed by phosphatases. Exergonic.

■ Hydrolysis of fructose-1,6-bisphosphate to produce fructose-6-phosphate and phosphate ion (∆G°́ = - 16.7 kJ/mol).

(1) Fructose-1,6-bisphosphatase - Allosteric enzyme: strongly inhibited by AMP, stimulated by ATP. - A control point in the pathway.

■ Hydrolysis of glucose-6-phosphate to glucose and phosphate ion (∆G°́ = - 13.8 kJ/mol).

(1) Glucose-6-phosphatase (2) Hydrolysis of glucose-6-phosphate to glucose occurs in the

endoplasmic reticulum.

III. Control of Carbohydrate Metabolism

1. Reciprocal regulation in glucose metabolism ■ Glycolysis–Gluconeogenesis: reciprocal regulated

■ An important element in the control process involves

fructose-2,6-bisphosphate (F2,6P). - An allosteric activator of phosphofructokinase (PFK) [glycolysis].

- An inhibitor of fructose bisphosphate phosphatase (FBPase) [gluconeogenesis].

- Concentration of F2,6P: high stimulates glycolysis, low stimulates gluconeogenesis.

■ The concentration of F2,6P in a cell depends on the balance between its synthesis [by phosphofructokinase-2 (PFK-2)], and its breakdown [by fructose-bisphosphatase-2 (FBPase-2)].

(1) The enzymes that control the formation and breakdown of F2,6P controlled by a phosphorylation/dephosphorylation mechanism.

(2) Both enzyme activities are located on the same protein (a dimer of about 100 kDa molecular mass).

(3) Phosphorylation increase in activity of FBPase-2; decrease in the concentration of F2,6P stimulating gluconeogenesis.

(4) Dephosphorylation increase in PFK-2 activity; increase in the concentration of F2,6P stimulating glycolysis.

■ The effect of fructose-2,6-bisphosphate on the activity of FBPase the inhibitor works by itself, but its

effect is greatly increased by the presence of the allosteric inhibitor AMP.

■ The final step of glycolysis is also a major control point in glucose metabolism (1) Pyruvate kinase (PK) is allosterically affected (Inhibitors: ATP and alanine). (2) Alanine is the amino version of pyruvate (by transaminase). A high level of alanine A high level of pyruvate Making pyruvate: shut down

(3) Fructose-1,6-bisphosphate allosterically activates PK so that the incoming

products of the first reactions of glycolysis can be processed.

(4) Pyruvate kinase is also found as isozymes (subunits: M, L, A). - M subunit, in muscle; L, in liver; A, in other tissues. - Liver isozymes subject to covalent modification and allosteric control. - Low levels of blood sugar trigger the release of glucagons production of a protein kinase.

■ Activation of glycogen phosphorylase: hormone-activated enzymatic cascade

2. Hormone control of glycogen metabolism

Important hormones: insulin, glucagon, epinephrine

■ Insulin

- In response to increased blood glucose levels secreted by the cells of the

islets of Langerhans in the pancreas

- Insulin binding to cell receptors

(1) Triggers protein kinase cascade leads to glycogen synthesis

(2) Triggers protein kinase cascade stimulates the action of GLUT4 (glucose

transport protein) released from vesicles cell surface brings

glucose into the cell glucose-6-phosphate (G-6-P)

(3) G-6-P two fates

i) After conversion to G-1-P incorporated into glycogen

ii) Serve as an allosteric activator of phosphorylated glycogen synthase

■ Glucagon (peptide hormone) and epinephrine (adrenaline, amino acid derivative)

- When blood glucose levels decrease,

glucagon and epinephrine secreted by the cells of the islets of Langerhans in

the pancreas

- Epinephrine: important on the very short time scale of the fight response - Glucagon: operates with insulin over a longer time scale to stabilize blood

glucose levels

3. General methods to regulate metabolism ■ Mechanisms of metabolic control

■ Substrate cycling - Opposing reactions can be catalyzed by different enzymes. - The opposing reactions can be independently regulated and have different

rates. [Example] (1) In glycolysis, the reaction catalyzed by phosphofructokinase is highly

exergonic under physiological conditions (∆G°́ = - 25.9 kJ/mol). Fructose-6-phosphate + ATP Fructose-1,6-bisphosphate + ADP

(2) The opposing reaction (gluconeogenesis) is also exergonic (∆G°́ = - 8.6 kJ/mol) and is catalyzed by fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphate + H2O Fructose-6-phosphate + Pi (3) The opposing reactions are not the exact reverse of one another.

two opposing reactions together: net reaction ATP + H2O ADP + Pi

Hydrolysis of ATP is the energetic price that is paid for independent control of the opposing reactions.

4. Glycogen metabolism in body organs

■ Using combinations of these control mechanisms, an organism can set

up a division of labor among tissues and organs to maintain control of glucose metabolism.

■ Clear example: Cori cycle - Fast-twitch muscle Comparatively few mitochondria Metabolism is largely anaerobic Produces lactate under conditions of oxygen debt.

- Liver Lactate is transported there by the blood Gluconeogenesis recycles

the lactate Glucose produced in the liver is transported back to skeletal muscle by the blood.

■ In the same cell (of whatever type), these two metabolic pathways (glycolysis and gluconeogenesis) are not highly active simultaneously.

IV. Glucose Is Sometimes Diverted through the Pentose Phosphate Pathway

■ Pentose phosphate pathway

- C5 sugars (including ribose) are produced from glucose.

- Production of nicotinamide adenine dinucleotide phosphate (NADPH).

(1) NADH is produced in the oxidative reactions

(2) NADPH is a reducing agent in biosynthesis

1. Pentose phosphate pathway: oxidative reactions

(1) Glucose-6-phosphate is oxidized to 6-phosphogluconate. - Enzyme: glucose-6-phosphate dehydrogenase. - NADPH is produced by the reaction.

(2) Oxidative decarboxylation, and NADPH is produced once again.

- 6-Phosphogluconate loses its carboxyl group (released as CO2), production of ribulose-5-phosphate.

- Enzyme: 6-phosphogluconate dehydrogenase.

2. Pentose phosphate pathway: nonoxidative reactions

■ In the remaining steps of the pentose phosphate pathway, several reactions

involve transfer of 2- and 3-carbon units.

■ Two different reactions in which ribulose-5-phosphate isomerizes.

(1) Phosphopentose-3-epimerase

- Ribulose-5-phosphate xylulose-5-phosphate (ketose).

(2) Phosphopentose isomerase

- Ribulose-5-phosphate ribose-5-phosphate (aldose).

■ Link the pentose phosphate pathway with glycolysis.

Group-transfer reactions require the two C5 sugars produced by the

isomerization of ribulose-5-phosphate.

(1) 2 molecules of X5P and 1 molecule of R5P rearrange to give 2

molecules of F6P and 1 molecule of glyceraldehyde-3-phosphate.

3 molecules of pentose (C5) give 2 molecules of hexose (C6) and

1 molecule of a triose (C3).

(2) Two enzymes, transketolase and transaldolase, are responsible for the

reshuffling of the carbon atoms of sugars.

■ Transketolase:

C2 unit from X5P (C5) is transferred to R5P (C5)

sedoheptulose-7-phosphate (C7) and glyceraldehyde-3-phosphate (C3).

■ Transaldolase:

C3 unit is transferred from sedoheptulose-7-phosphate (C7) to the C3

glyceraldehyde-3-phosphate F6P (C6) and erythrose-4-phosphate (C4).

■ Transketolase:

X5P reacts with E4P F6P and glyceraldehyde-3-phosphate.

3. Pentose phosphate pathway: regulation

■ The reactions catalyzed by transketolase and transaldolase are reversible respond to the needs of an organism. (1) If the organism needs more NADPH than R5P the reaction series goes through the complete pathway just discussed.

6 G6P + 12NADP+ + 6H2O 6 R5P + 6CO2 + 12NADPH + 12H+

(2) If the organism has a greater need for R5P than for NADPH F6P and glyceraldehyde-3-phosphate can give rise to R5P by the

successsive operation of the transketolase and transaldolase reactions, bypassing the oxidative portion of the pentose phosphate pathway (follow the red shaded path down to glyceraldehyde-3-phosphate and then go up to R5P).