Glucose is a major metabolic fuel. Organisms store excess compounds such glucose in readily available storage compound - glycogen. In plants the carbohydrate storage compound is starch. The branching in the chemical structure of glycogen provides a means for rapid degradation and release of energy.
1. Muscles cannot mobilize fat as rapidly as they can glycogen.
2. The fatty acid residues of fat cannot be metabolized anaerobically.
3. Animals cannot convert fatty acids to glucose, so fat metabolism alone cannot adequately maintain blood glucose levels for brain fuel.
I. Glycogen Breakdown
Liver and muscle store glycogen. Muscle needs ATP for movement and converts glycogen to glucose-6-phosphate for glycolysis. In liver, low blood [glucose] triggers glycogen breakdown to G6P that is hydrolyzed to glucose and supplied to the blood stream.
Glycogen breakdown requires three enzymes
1. Glycogen phosphorylase
Glycogen + Pi <===> glycogen -1 + G1P
requires 5 residues on a branch
2. Glycogen debranching enzyme removes glycogen branches
90% of glycogen converted to G1P, 10% to glucose
G1P <---> G6P
A. Glycogen Phosphorylase
Glycogen phosphorylase is a dimer of 842-amino acid residues (97-kD) that catalyzes the rate-controlling step in glycogen breakdown. It exists in two forms.
a phosphoryl esterified to ser 14 in each subunit
Structural Domains and Binding Sites
X-ray structure known for both phosphorylase a and b. (Fletterick & Johnson)
Interface subdomain, 1-315
Ser 14 that is phosphorylated
Allosteric effector site
the intersubunit contacts in the dimer
a glycogen-binding subdomain, 316-484
glycogen forms a left-handed helix with 6.5 glucose residues
a 30 angstrom crevice has the same radius of curvature as glycogen; the crevice can contain 4-5 glucose residues but not a branched glycogen
Pyridoxal phosphate is an essential cofactor for phosphorylase
Covalently linked via a Schiff base with lys 680.
Can be reduced and still function.
Only the phosphate group functions in catalysis. Probably as an acid-base catalyst.
Kinetics and reaction mechanism
Cleave C1-O1 bond to yield G1P.
See Fig. 17-4 for the mechanism.
Oxonium ion formation
1,5-gluconolactone is a potent inhibitor.
Rabbit muscle phosphoglucomutase has a largely buried active site within a 561-residue monomer. Phosphoglucomutase is similar to that caused by phosphoglycerate mutase. A phosphoryl group is transferred from the active phosphoenzyme to G1P, forming glucose 1,6-bisphosphate. Then the enzyme is rephosphorylated while the phosphoryl group on the glucose is shifted between the C1 and C6 position
Phosphoglucokinase catalyzes the formation of G 1,6bisphosphate to replenish that which is lost upon dissociation from the enzyme.
C. Glycogen Debranching Enzyme
Glycogen debranching enzyme acts as an a (1-->4) transglycosylase (glycosyl transferase). The debranching enzyme has active sites for both the transferase reaction and the a (1-->4) transglycosylase reaction.
D. Thermodynamics of Glycogen Metabolism:
The Need for Separate Synthetic and Degradative Pathways
DG for the phosphorylase reaction is + 3.1 kJ/mol for standard conditions, but with cellular concentrations the DG range is -5 to -8 kJ/mol. Under physiological conditions, glycogen breakdown is exergonic. The synthesis of glycogen from G1P under physiological conditions is unfavorable. Glycogen biosynthesis and breakdown occur by separate pathways.
II. Glycogen Synthesis
McArdle's disease - painful muscle cramps - no glycogen phosphorylase. Can't break glycogen down, but glycogen is present.
UDP-glucose formation enables the donation of glycosyl groups to growing glycogen chains.
Three enzymes for glycogen synthesis:
A. UDP-Glucose Pyrophosphorylase
UTP + G1P ---> UDPG + PPi is catalyzed by UDP-glucose pyrophosphorylase. Hydrolysis of the pyrophosphate makes the overall reaction highly exergonic. DG -33.5 kJ/mol
B. Glycogen Synthase
Glycogen synthase DG is -13.4 kJ/mol. Two glucoses can't be linked together in the first step; there is addition of a glycosyl residue to a growing glycogen chain. A protein called glycogenin is the starting point with tyrosine 194 being glycosylated then there is autocatalytic addition of up to 7 additional glycosyl groups to form a glycogen primer. Each glycogen granule contains one glycogenin.
C. Glycogen Branching
A separate enzyme amyl-(1,4-->1,6) transglycosylase is the branching enzyme. it is distinct from the debranching enzyme.
III. Control of Glycogen Metabolism
Both synthesis and breakdown of glycogen are exergonic under physiological conditions. What prevents a futile cycle? Glycogen phosphorylase and glycogen synthase are oppositely controlled by covalent modification.
A. Direct Allosteric Control of Glycogen Phosphorylase and Glycogen Synthase
The active R form phosphorylase b is converted to an inactive T form by an allosteric interaction of ATP and/or G6P phosphorylation. The inactive T form is covalently modified using phosphorylase kinase and ATP. This reaction is opposed by phosphoprotein phosphatase. AMP stimulates the conformation shift from T form back to the active R form. A high level of glucose converts phosphorylase a R form to the T form that is subject to activation using phosphoprotein phosphatase.
B. Covalent Modification of Enzymes by Cyclic Cascades: Effector "Signal" Amplification
Glycogen synthase and glycogen phosphorylase can each be enzymatically interconverted between two forms with different kinetic and allosteric properties through a complex series of cyclic cascade reactions. The interconversion of these different enzyme forms involves distinct enzyme-catalyzed covalent modification and demodification reactions.
Enzymatically interconvertible enzymes systems
1. Can respond to a greater number of allosteric stimuli.
2. Exhibit greater flexibility in their control patterns.
3. Possess enormous amplification potential in their responses to variations in effector concentrations.
This is because the modifying and demodifying enzymes themselves are subjected to allosteric control. There are regulators that regulate the regulators that regulate the regulators that control the reaction. See F 17-11 and F 17-12 for mono and bicyclic cascades.
Description of a General Cyclic Cascade
In the steady state, the fraction of E in the active form is
(where [E]T = [Ea] + [Eb] is the total enzyme concentration)
determines the rate of the reaction catalyzed by E.
This fraction is a function of the of the modifying enzyme [F]T and [R]T,
the concentration of their allosteric effectors e1 and e2,
the dissociation constants of these effectors, K1 and K2, and
the substrate dissociation constants , Kf and Kr, of the target enzymes,
as well as the rate constants, kf and kr, for the interconversions themselves.
A complex relationship. But a small change in the concentration of e1 can cause a large change in the fraction of enzyme in an active form. This is an amplification. The cascade function to amplify the sensitivity of the system to an allosteric effector.
A bicyclic cascade increase the amplification.
The activities of both glycogen phosphorylase and glycogen synthase are controlled by bicyclic cascades.
C. Glycogen Phosphorylase Bicyclic Cascade
In 1938, Carl & Gerti Cori found that glycogen phosphorylase existed in two forms,
the b form required AMP for activity, and
the a form that is active without AMP.
In 1959, Ed Krebs & Ed Fischer demonstrated that in
the a form Ser 14 is phosphorylated, and
the b form there is no modification.
Glycogen Phosphorylase: The Cascade's Target Enzyme
Glycogen phosphorylase activity is allosterically controlled
AMP activation and
ATP, G6P, and glucose inhibition.
Superimposed on this allosteric control is covalent modification/demodification.
1. Phosphorylase kinase
phosphorylates Ser 14 of glycogen phosphorylase b
2. cAMP-dependent protein kinase
phosphorylates glycogen phosphorylase kinase
3. Phosphoprotein phosphatase-1
glycogen phosphorylase a
glycogen phosphorylase kinase
deactivates both enzymes.
Phosphorylation of Ser 14 shifts the enzyme's T (inactive) <====> R (active) 14 equilibrium in favor of the R state. The phosphate on Ser 14 is analogous to an allosteric effector..
In resting cells [ATP] and [G6P] are high enough to inhibit phosphorylase b. The activity is determined by the fraction of enzyme present as phosphorylase a.
the amount of phosphorylated enzyme (Ea) depends on the relative activities of phosphorylase kinase and phosphoprotein phosphatase.
cAMP-Dependent Protein Kinase: A Crucial Regulatory Link
converts phosphorylase b to phosphorylase a
is subject to covalent modification/demodification.
To be fully active Ca2+ must present and the enzyme must be phosphorylated.
cAMP is the second messenger that regulated both cascades.
cAMP is made by adenylate cyclase and destroyed by phosphodiesterase
cAMP-dependent protein kinase is shown in F 17-14.
Phosphorylase Kinase: Coordination of Enzyme Activation with [Ca2+].
Phosphorylase kinase is activated by [Ca2+] as low as 10-7 M as well as by covalent modification.
Calmodulin: A Ca2+-Activated Switch.
The binding of Ca2+-CaM to myosin light chain kinase (MLCK) activates the enzyme.
The [cAMP] controls the fraction of an enzyme in its phosphorylated form, not only by increasing the rate of phosphorylation, but also by decreasing the rate at which it is dephosphorylated (a double input). In the case of glycogen phosphorylase, an increase in [cAMP] increases rate of activation and decreases rate of inactivation.
Both the R and T forms of phosphorylase a strongly bind phosphoprotein phosphatase-1, but only in the T state enzyme is the Ser 14-phosphoryl group accessible for hydrolysis. When phosphorylase a is in its active R form it removes phosphoprotein phosphatase-1 from circulation.
See F 17-18
D. Glycogen Synthase Bicyclic Cascade
Like glycogen phosphorylase, glycogen synthase exists in two interconvertible forms.
1. The modified form (m; phosphorylated) form is inactive under physiological conditions.
2. The original (o; dephosphorylated) form is active.
The other side of the coin - oppositely regulated.
E. Integration of Glycogen Metabolism Control Mechanisms
Whether there is net synthesis or degradation of glycogen and what rate will depend on the relative balance of the active forms of glycogen synthase and glycogen phosphorylase.
This in turn, largely depends on the rates of phosphorylation/dephosphorylation of the two bicyclic cascades.
The bicyclic cascades are linked by
cAMP-dependent protein kinase
that are activated
in turn glycogen phosphorylase is activated and glycogen synthase is inactivated.
Also linked by
which is inhibited by phosphorylase a
is not able to activate glycogen synthase
until it first inactivates phosphorylase a.
Hormones Trigger Glycogen Metabolism through the Intermediacy of Second Messengers
The adenylate cyclase makes a second messenger.
The production is controlled via hormone receptors.
Glucagon and epinephrine stimulate.
F. Maintenance of Blood Glucose Levels
An important function of the liver is to maintain the blood concentration of glucose, the brain's primary fuel so that you can think about the wonders of biochemistry. The body tries to maintain a level of ~ 5 mM; 100 mg/dl.
A decrease in blood glucose - after exercise
1. stimulates the release of glucagon from the a-cells of the pancreas,
2. glucagon receptors on liver cells bind glucagon thus activating adenylate cyclase that increase [cAMP]
3. [cAMP] increase triggers the breakdown of glycogen makes G6P,
4. G6P is hydrolyzed by glucose-6-phosphatase to make glucose. Now available to replenish blood glucose and give pleasant thoughts of the wonders of biochemistry.
The b-cells of the pancreas sense the [glucose] and secrete insulin that stimulates cells to remove blood glucose.
Glucokinase Forms G6P at a Rate Proportional to the [Glucose]
The liver "buffer" blood [glucose] using glucokinase. See handout on the glucose sensor.
There is a glucokinase regulatory protein that is a competitive inhibit when F6P is also present.
Fructose-2,6-bisphosphate Activates Glycolysis
F2,6P is not a glycolytic intermediate. It is an extremely potent allosteric activation of animal PFK and an inhibitor of FBPase. My friend Ko Uyeda was one of the codiscoverers.
The [F2,6P] depends on the balance between
rate of synthesis
rate of degradation
These enzymes are located in different domains of a ~100 kD homodimeric protein and are subject to allosteric regulation by a variety of metabolic intermediates and by covalent modification/demodification.
Tissue specificity of isozyme allows heart and skeletal muscle to behave differently from liver.
G. Response to Stress
Epineprine, norepineprine (adrenaline)
fight or flee
See F 17-22
IV. Glycogen Storage Diseases
There are nine types
see Table 17-1
the enzyme deficiency
Prepared 2/12/97 FRL