Glycogen Metabolism

Complex carbohydrates

Oligosaccharides Polysaccharides

Starch Glycogen

Starch

Major storage carbohydrate in higher plants Amylose long straight glucose chains (a1-4) Amylopectin branched every 4-!" glc residues (a 1-#) $rovides %"& o' dietary calories in humans (orld(ide

GLYCOGEN METABOLISM

Glycogen: a highly branched polymer of glucose. Chains have glycosidic links 1 4. Branches are linked 1 6.

Glycogen
G G G G G G G G G G G G G G G G G

Major storage carbohydrate in animals )ong straight glucose chains (a1-4) G G *ranched every 4-% glc residues (a 1-#) More branched than 1-4 link starch )ess osmotic pressure +asily mobili,ed

1-6 link

CH2OH H O H CH2OH O H 1 H O OH O H H 4 OH H H H H 2 OH O OH O CH2OH O H OH H H OH OH H H OH H 1 O H

CH2OH

H OH

glycogen

OH

H O H H O 3 H 4 H OH

OH

CH2OH

CH2OH

H OH

OH

H OH

6 CH2 5 H OH

OH

Glycogen is a polymer of glucose residues linked by (1 4) glycosidic bonds, mainly (1 6) glycosidic bonds, at branch points. Glycogen chains & branches are longer than shown.

Glucose is stored as glycogen predominantly in liver and muscle cells.

Glucose stored in polymeric form as glycogen mostly in the liver and skeletal muscle.

Glucose can be rapidly delivered to blood stream when needed upon degradation of glycogen. = glycogenolysis

nough glucose and energy triggers synthesis of glycogen. = glycogenesis

-- GLYCOGENOLYSIS-DEGRADATION OF GLYCOGEN

1. !elease of glucose"1"phosphate n#yme = glycogen phosphorylase non-

re !cing en s

"

#i

gl!cose-$- " phospha%e

CH2OH H H OH H OPO32 OH H OH O H

Glycogen catabolism (breakdown):

glucose-1-phosphate

Glycogen Phosphorylase catalyzes phosphorolytic cleavage of the (1 4) glycosidic linkages of glycogen, releasing glucose-1-phosphate as reaction product.

glycogen(n residues) + Pi glycogen (n1 residues) + glucose-1-phosphate

This phosphorolysis may be compared to hydrolysis: R-OH + R'-OH Hydrolysis: R-O-R' + HOH Phosphorolysis: R-O-R' + HO-PO32R-OH + R'-O-PO32-

H O P O O + N H H2 C O C

O OH

Pyridoxal phosphate (PLP), a derivative of vitamin B6, serves as prosthetic group for Glycogen Phosphorylase.

CH3

pyridoxal phosphate (PLP)

lysine
(CH2)4 N+ O P O + N H O CH3 H2 C O HC O H
COO

Enz

H3N+

CH2

CH2

CH2

CH2

NH3

Enzyme (Lys)-PLP Schiff base

Pyridoxal phosphate (PLP) is held at the active site by a Schiff base linkage, formed by reaction of the aldehyde of PLP with the -amino group of a lysine residue.

In contrast to its role in other enzymes, the phosphate of PLP is involved in acid/base catalysis by Phosphorylase.

Enz (CH2)4 N+ O P O O + N H CH3 H2 C O HC H O

The Pi substrate binds between the phosphate of PLP and the glycosidic O linking the terminal glucose residue of the glycogen.

Enzyme (Lys)-PLP Schiff base

After the phosphate substrate donates H+ during cleavage of the glycosidic bond, it receives H+ from the phosphate moiety of PLP.

PLP then takes back the H+ as the phosphate O attacks C1 of the cleaved glucose to yield glucose-1-phosphate.

PLP

GlcNAc

inhibitor

Glycogen Phosphorylase: a homodimeric enzyme, subject to allosteric control. It transitions between relaxed (active) & tense (inhibited) conformations.
GlcNAc PLP Human Liver Glycogen Phosphorylase
PDB 1EM6

A glucose analog, N-acetylglucosamine (GlcNAc), is adjacent to pyridoxal phosphate at the active site in the crystal structure shown.

GlcNAc

inhibitor

PLP

A class of drugs developed for treating PLP the hyperglycemia of diabetes (chloroindolecarboxamides), inhibit liver Phosphorylase allosterically. These inhibitors bind GlcNAc at the dimer interface, stabilizing the inactive Human Liver (tense) conformation. Glycogen Phosphorylase

PDB 1EM6

A glycogen storage site on the surface of the Phosphorylase enzyme binds the glycogen particle.

Given the distance between storage & active sites, Phosphorylase can cleave (1 4) linkages only to within 4 residues of an (1 6) branch point.

This is called a "limit branch".

Debranching enzyme has 2 independent active sites, consisting of residues in different segments of a single polypeptide chain: The transferase of the debranching enzyme transfers 3 glucose residues from a 4-residue limit branch to the end of another branch, diminishing the limit branch to a single glucose residue. The (1 6) glucosidase moiety of the debranching enzyme then catalyzes hydrolysis of the (1 6) linkage, yielding free glucose. This is a minor fraction of glucose released from glycogen.

The major product of glycogen breakdown is glucose-1-phosphate, from Phosphorylase activity.

$ebranching " two parts n#yme = e&ranching en'y(e %both& %1 6& link

transferase

'ransfers chain of three glucoses to any nonreducing end

 %1 6& link debranching en#yme %glucosidase&

"
= glucose

1(6 linkage cleaved

Enzyme-Ser-OPO32 Enzyme-Ser-OH CH2OPO32 H H OH H OPO32 OH H OH OH H H OH H OH OH O H H O H CH2OPO32

Enzyme-Ser-OPO32

CH2OH

H OH

OH

OPO32

OH

glucose-1-phosphate

glucose-6-phosphate

Phosphoglucomutase catalyzes the reversible reaction: glucose-1-phosphate glucose-6-phosphate

A serine OH at the active site donates & accepts Pi. The bisphosphate is not released. Phosphoglycerate Mutase has a similar mechanism, but instead uses His for Pi transfer.

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

Glucose-6-phosphate may enter Glycolysis or (mainly in liver) be dephosphorylated for release to the blood. Liver Glucose-6-phosphatase catalyzes the following, essential to the liver's role in maintaining blood glucose: glucose-6-phosphate + H2O glucose + Pi

Most other tissues lack this enzyme.

O CH2OH HN O H O O H OH O

H H OH H P O

Glycogen synthesis
O O H H OH P O CH2 O O OH

H H OH

UDP-glucose

Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis. As glucose residues are added to glycogen, UDP-glucose is the substrate and UDP is released as a reaction product. Nucleotide diphosphate sugars are precursors also for synthesis of other complex carbohydrates, including oligosaccharide chains of glycoproteins, etc.

UDP-Glucose Pyrophosphorylase
HN H O O O O O O P O O O O P O P O CH2 H H OH O O N

CH2OH

H OH

OH

+
O P

O H H OH

OH

glucose-1-phosphate
PPi
HN H O O O

UTP
O

CH2OH O O

H O O P O CH2

H OH P

OH

O H H OH H H OH

OH

UDP-glucose

UDP-glucose is formed from glucose-1-phosphate: glucose-1-phosphate + UTP UDP-glucose + PPi PPi + H2O 2 Pi UDP-glucose +

Overall: glucose-1-phosphate + UTP 2 Pi

Spontaneous hydrolysis of the ~P bond in PPi (P~P) drives the overall reaction.

Cleavage of PPi is the only energy cost for glycogen synthesis (one ~P bond per glucose residue).

Glycogenin initiates glycogen synthesis.

Glycogenin is an enzyme that catalyzes attachment of a glucose molecule to one of its own tyrosine residues.

Glycogenin is a dimer, and evidence indicates that the 2 copies of the enzyme glucosylate one another.

Tyr active site

active site Tyr

Glycogenin dimer

6 CH

2OH

H O C O Uridine HO C CH H2 NH O O O
6 CH

O O O P O

UDP-glucose

H P

tyrosine residue of Glycogenin

H OH

OH

OH

2OH

O H H OH H
1

O-linked glucose H residue 4

C O
2 3

OH H OH

C CH H2 NH

+ UDP

A glycosidic bond is formed between the anomeric C1 of the glucose moiety derived from UDP-glucose and the hydroxyl oxygen of a tyrosine side-chain of Glycogenin. UDP is released as a product.

6 CH

2OH

O H C
1

O-linked glucose H residue 4

H OH H O
2

OH
3

UDP-glucose
CH2OH H H OH H O H OH O H O H C C CH H2 NH O

OH

C CH H2 NH

+ UDP

CH2OH

H OH

OH

+ UDP

OH

(1 4) linkage

Glycogenin then catalyzes glucosylation at C4 of the attached glucose (UDP-glucose again the donor), to yield an O-linked disaccharide with (14) glycosidic linkage. This is repeated until a short linear glucose polymer with (14) glycosidic linkages is built up on Glycogenin.

Glycogen Synthase then catalyzes elongation of glycogen chains initiated by Glycogenin.

)" Glycogen synthesis n#yme = glycogen syn%hase

)D#"*gl!cose+n"$

)D#-gl!cose " *gl!cose+n

glycogen

glucose always added to nonreducing end. 'he glycosidic bond formed is (1 4). glycogen synthase is inhibited by phosphorylation( regulated by glucagon % discussed later&

Glycogen Synthase catalyzes transfer of the glucose moiety of UDP-glucose to the hydroxyl at C4 of the terminal residue of a glycogen chain to form an (1 4) glycosidic linkage:

glycogen(n residues) + UDP-glucose glycogen(n +1 residues) + UDP

A branching enzyme transfers a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with an (16) linkage.

branching en'y(e

introduces branching by transferring a teminal fragment of 6-7 residues from a growing chain to a 6-position farther back in the chain. makes a branch with an (1 6) link and two ends to add glucose. branching accelerates the rate of glucose release during degradation.

branching enzy e

new 1,6 bond

Me%a&olic Reg!la%ion o, Ma((alian Glycogen Le-els

"" Glycogen reserves are the most immediately available large source of metabolic energy for mammals.

"" *torage and utili#ation are under dietary and hormonal control.

+rimary hormones = -- epinephrine %adrenaline& = ,fight"or"flight"" glucagon -- insulin

+rimary en#yme targets in glycogen metabolism= glycogen phosphorylase and glycogen synthase. 'he actions of the hormones are indirect.

!"a ple- hormones and diet

$inner at ./00 pm "" steak( mashed potatoes( dessert( wine

*leep immediately( sleep late

$uring sleep/ amino acids( C123 high blood glucose levels higher insulin glycogenosynthesis

4ake late for class adrenaline rush run to class glycogen glucose lactate epinephrine %= adrenaline& glycogenolysis

........ #$%&$'!( ........... Glucagon - low glucose le)els "" 5cts primarily in li-er. "" 5 polypeptide hormone produced in "cells of the islets of 6angerhan of the pancreas. "" !eceptors on surface of liver cells. "" *timulates glycogen breakdown 7 inhibits glycogenesis. "" Glucagon also blocks glycolysis 7 stimulates gluconeogenesis.

!pinephrine - low glucose le)els

"" 5cts primarily on skeletal (!scle. "" !eceptors on surface of cells. "" *timulates glycogen breakdown 7 inhibits glycogenesis.

Glucagon and epinephrine both stimulate intracellular pathway via increasing levels of c58+.

*nsulin

"" #igh le)els of glucose induce release of insulin from "cells of islets of 6angerhan in the pancreas. "" 9nsulin is polypeptide hormone. "" $etected by receptors at surface of uscle cells. "" 9ncreases glycogenesis in muscle. :act intracellular pathway not yet known but target of insulin is known.

Glycogen Synthesis UTP UDP + 2 Pi glycogen(n + 1) Pi

glycogen(n) + glucose-1-P Glycogen Phosphorylase

Both synthesis & breakdown of glycogen are spontaneous. If both pathways were active simultaneously in a cell, there would be a "futile cycle" with cleavage of one ~P bond per cycle (in forming UDP-glucose). To prevent such a futile cycle, Glycogen Synthase and Glycogen Phosphorylase are reciprocally regulated, by allosteric effectors and by phosphorylation.

Glycogen Phosphorylase in muscle is subject to allosteric regulation by AMP, ATP, and glucose-6phosphate.

A separate isozyme of Phosphorylase expressed in liver is less sensitive to these allosteric controls.

AMP (present significantly when ATP is depleted) activates Phosphorylase, promoting the relaxed conformation.

ATP & glucose-6-phosphate, which both have binding sites that overlap that of AMP, inhibit Phosphorylase, promoting the tense conformation.

Thus glycogen breakdown is inhibited when ATP and glucose-6-phosphate are plentiful.

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

Glycogen Synthase is allosterically activated by glucose-6-P (opposite of effect on Phosphorylase).

Thus Glycogen Synthase is active when high blood glucose leads to elevated intracellular glucose-6-P.

It is useful to a cell to store glucose as glycogen when the input to Glycolysis (glucose-6-P), and the main product of Glycolysis (ATP), are adequate.

Regulation by covalent modification (phosphorylation):

The hormones glucagon and epinephrine activate G-protein coupled receptors to trigger cAMP cascades.

Both hormones are produced in response to low blood sugar.

Glucagon, which is synthesized by -cells of the pancreas, activates cAMP formation in liver.

Epinephrine activates cAMP formation in muscle.

c+&, -ascade

"" 5 cyclic 58+ cascade is used by both epinephrine and glucagon. "" 5 cascade is a mechanism in which en#ymes activate other en#ymes se;uentially usually leading to an amplification of an initial signal.

The cAMP cascade results in phosphorylation of a serine hydroxyl of Glycogen Phosphorylase, which promotes transition to the active (relaxed) state.

The phosphorylated enzyme is less sensitive to allosteric inhibitors.

Thus, even if cellular ATP & glucose-6-phosphate are high, Phosphorylase will be active.

The glucose-1-phosphate produced from glycogen in liver may be converted to free glucose for release to the blood.

With this hormone-activated regulation, the needs of the organism take precedence over needs of the cell.

Commonly used terminology:

"a" is the form of the enzyme that tends to be active, and independent of allosteric regulators (in the case of Glycogen Phosphorylase, when phosphorylated).

"b" is the form of the enzyme that is dependent on local allosteric controls (in the case of Glycogen Phosphorylase when dephosphorylated).

Hormone (epinephrine or glucagon) via G Protein (G-GTP) Adenylate cyclase (inactive) ATP Activation AMP Protein kinase A (inactive) Protein kinase A (active) ATP ADP Phosphorylase kinase (b-inactive) Phosphatase Pi Phosphorylase (b-allosteric) Phosphatase Pi Phosphorylase kinase (P) (a-active) ATP ADP Phosphorylase (P) (a-active) cyclic AMP + PPi Phosphodiesterase Adenylate cyclase (active) catalysis

Signal cascade by which Glycogen Phosphorylase is activated.

Epinephrine/Gl!cagon Casca e !egulating Glycogen 8etabolism

Epinephrine AC

O!%si e

Recep%or G#ro%ein Insi e

#M c58+<++i

5'+

5C = adenylate cyclase

+8 = plasma membrane

The cAMP cascade induced in liver by glucagon or epinephrine has the opposite effect on glycogen synthesis.

Glycogen Synthase is phosphorylated by Protein Kinase A as well as by Phosphorylase Kinase.

Phosphorylation of Glycogen Synthase promotes the "b" (less active) conformation.

The cAMP cascade thus inhibits glycogen synthesis.

Instead of being converted to glycogen, glucose1-P in liver may be converted to glucose-6-P, and dephosphorylated for release to the blood.

Reversible covalent modification

Glycogen synthesis and degradation as main example enzymes effect of phosphorylation control by hormones

Glycogen synthesis and degradation

glycogen
Glycogen synthase UDP-glucose

Glycogen phosphorylase

Glucose 1 P

glycogen (n-1)

Different enzymes for forward and reverse reactions. This allows reciprocal regulation (no futile cycles).

Glycogen synthesis and degradation

glycogen
Phosphorylation inactivates GS
Glycogen synthase

Phosphorylation activates GP Glycogen

phosphorylase

Glucose 1 P

UDP-glucose

glycogen (n-1)

Phosphorylation has opposite effects on synthesis and degradation.

Glycogen phosphorylase
ATP
kinase phosphatase

GP

GP- P

Glycogen phosphorylase is a dimer: 2 x 97 kDa Each monomer can be phosphorylated at Ser 14, making the enzyme more active.

GP

Structural changes

Ser 14 phosphorylated (right) causes changes at the dimer interface. Buchbinder et al Biochemistry 36:8039-44, 1997

Glycogen phosphorylase
Dimer
Glucose unit is red; Ser 14 is yellow; pyridoxal phosphate is pale blue and AMP (allosteric effector) is dark blue.
Lehninger 8-29

The kinases

Some are quite general and phosphorylate lots of proteins.

Glycogen phosphorylase is phosphorylated by its own kinase: GP kinase.

It, in turn, is activated by PKA

Protein kinase A (PKA)

Allosteric regulation of glycogen phosphorylase ATP

kinase phosphatase

GP

GP- P
active

Less active

allosteric reg + by AMP - by G6P and ATP

Less active form is also allosterically-regulated

interplay between: reversible covalent modification allosteric regulation

Balance of glycogen phosphorylase forms

Can be less active or active Less active form can also be R/T

Relatively high [ATP] and [G6P] will keep less active form mostly in T form (2 systems switch off)

Active form is mostly in R form

Glycogen synthase

ATP
kinase

GS

phosphatase

GS- P

GS is a tetramer: 4 x 85 kDa each monomer can be phosphorylated on up to 9 different Ser residues by different kinases. Phosphorylation makes less active.

Glycogen synthesis and degradation

glycogen
Phosphorylation inactivates GS
Glycogen synthase

Phosphorylation activates GP Glycogen

phosphorylase

Glucose 1 P

UDP-glucose

glycogen (n-1)

Phosphorylation drives degradation.

Allosteric regulation of glycogen synthase

ATP kinase phosphatase less active allosteric reg + G6P GS- P

GS

active

Less active form is also allosterically-regulated

interplay between: reversible covalent modification allosteric regulation

Adrenalin or glucagon

signal need for energy

Each acts through G-protein coupled receptor and activates adenyl cyclase cAMP

cAMP-dependent kinase phosphorylates key enzymes in metabolism and leads to glycogen DEGRADATION.

glycogen phosphorylase becomes active glycogen synthase becomes less active

Insulin
synthesis

signals well-fed state

Acts through Tyr-kinase receptor

Leads to dephosphorylation of key enzymes in metabolism and leads to glycogen SYNTHESIS.

glycogen synthase becomes active glycogen phosphorylase becomes less active

Glycogen synthesis and degradation

glycogen
Glycogen synthase UDP-glucose

Glycogen phosphorylase

Glucose 1 P

glycogen (n-1)

Phosphorylation drives degradation so switched on by adrenalin and glucagon, but switched off by insulin.

Hormonal control of metabolism

Hormones dictate phosphorylation state of enzymes General rule on synthesis/breakdown: synthesis

(insulin drives)

dephosphorylation

phosphorylation

catabolism (adrenalin drives)

Note: Reciprocal regulation Amplification Delicate control

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

High cytosolic glucose-6-phosphate, which would result when blood glucose is high, turns off the signal with regard to glycogen synthesis.

The conformation of Glycogen Synthase induced by the allosteric activator glucose-6-phosphate is susceptible to dephosphorylation by Protein Phosphatase.

Insulin, produced in response to high blood glucose, triggers a separate signal cascade that leads to activation of Phosphoprotein Phosphatase.

This phosphatase catalyzes removal of regulatory phosphate residues from Phosphorylase, Phosphorylase Kinase, & Glycogen Synthase enzymes.

Thus insulin antagonizes effects of the cAMP cascade induced by glucagon & epinephrine.

Ca++ also regulates glycogen breakdown in muscle.

During activation of contraction in skeletal muscle, sarcoplasmic reticulum Ca++-release channels open.

Ca++ released to the cytosol activates actin/myosin interactions.

Phosphorylase Kinase partly active fully active

inactive

Phosphorylase Kinase-Ca++

P-Phosphorylase Kinase-Ca++

Phosphorylase Kinase in muscle includes calmodulin as its subunit. Phosphorylase Kinase is partly activated by binding of Ca++ to this subunit. Phosphorylation of the enzyme, via a cAMP cascade induced by epinephrine, results in further activation. These regulatory processes ensure release of phosphorylated glucose from glycogen, for entry into Glycolysis to provide ATP needed for muscle contraction.

Nicotine

Major alkaloid in the genus Nicotiana

Nicotiana, a large genus mainly from New World (~ 65 species) in the Solanaceae Cultivated species: N. tabacum & N. rustica

Nicotiana tabacum

Nicotine

Nicotine effects and mode of action

Acts as a CNS stimulant and also affects peripheral nervous system Stimulation of adrenal glands and discharge of epinephrine (adrenaline) Nicotine also suppresses insulin output from the pancreas, which means that smokers are always slightly hyperglycemic In addition, nicotine indirectly causes a release of dopamine in the reward region of brain that controls pleasure and motivation

Mode of action on brain

Nicotine exerts its effects in the brain by acting on a specific type of receptor for acetylcholine, known as the nicotinic receptor

These receptors on dopamine neurons within the ventral tegmental area Activates acetylcholine receptors (locks the channel open) - this increases the activity of these dopamine neurons, leading to an increase in dopamine release in synapse, which is thought to mediate reward

Chemical Messengers in the NS

Neurotransmitters

Endorphins

Hormones

Neurotransmitters

Neurotransmitters travel from one neuron to another. Changes occur in the receiving neurons membrane, The ultimate effect is either:

Excitatory: the probability that the receiving neuron will fire increases Inhibitory: the probability that the receiving neuron will fire decreases

Neurotransmitters

Serotonin Sleep, appetite, sensory perception, temperature regulation, pain suppression, and mood Dopamine Voluntary movement, learning, memory, and emotion Acetylcholine Muscle action, cognitive functioning, memory, and emotion

Neurotransmitters

Norepinephrine Increased heart rate and the slowing of intestinal activity during stress, learning, memory, dreaming, waking from sleep, and emotion GABA (gama-aminobutyic acid) The major inhibitory neurotransmitter in the brain

Hormones

Insulin Produced by the pancreas Regulates the bodys use of glucose & affects appetite

Melatonin Secreted by the pineal gland Helps to regulate daily biological rhythms and promotes sleep.

Hormones

Adrenal Hormones Produced by the adrenal glands are involved in emotion and stress. They rise in response to nonemotional conditions, such as cold, heat, pin injury, and physical exercise, and in response to some drugs such as caffeine and nicotine. The Outer Part Cortisol The Inner Part Epinephrine & Norepinephrine

Hormones

Sex Hormones Are secreted by the gonads and by the adrenal glands Androgens Masculinizing Hormones Estrogens Feminizing Hormones

The Endocrine System

The Endocrine System

The bodys slow chemical communication system; a set of glands that secrete hormones into the blood stream.

The Endocrine System A Set of Glands

The Pituitary Gland A sort of master gland It is cherry-sized endocrine gland The hormones it secretes affect growth and the secretion of other endocrine glands The real boss is the hypothalamus Feedback System brain pituitary other glands hormones brain

Lateralization
Right Hemisphere Nonverbal areas Comprehension, spatial relationships, drawing, music, emotion Processes info. As a whole intuitive

Left Hemisphere Verbal competence Speaking, reading, thinking & reasoning Processes info in sequence One piece of data at a time logical

Emotion and Lateralization

Right Hemisphere Important for the expression of negative emotion Damage to the R.H. may make people euphoric. Activation in the R.H. leads to tendencies to withdraw from people.

Left Hemisphere Important for the expression of positive emotion Damage to the L.H. leads to loss of the capacity of joy. Activation in the L.H. leads to tendencies to approach other people.