chapter 21: glycogen metabolism copyright © 2007 by w. h. freeman and company berg tymoczko stryer...

Chapter 21:Glycogen Metabolism

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Glycogen Structure

A branched polymer of glucose with no phosphates.Chains are -1,4 and branch points are -1,6.One reducing end and many non-reducing ends.

MW 250000 to > 1000000

Glycogen Structure

Reducing end

Branch points every 8-10 residues.

Relationship to other pathways

Glycogenolysis

Attack is at the non-reducing ends of glycogen. Cleavage is catalyzed by phosphorylase. The reaction proceeds through a carbocation intermediate and uses pyridoxal phosphate (PLP) in the role of a conjugate acid conjugate base.

Incorporation of Pi

Keq for the reverse of the reaction below is ~3.6 which reflects the ratio of Pi/G-1-P.

Go' = -RT ln 3.6 = -3.3 kJ/mol at 37o

This Go' would lead to the conclusion that this reaction could be used in making glycogen.However, even with Pi/G-1-P = 300 glycogen synthesis occurs.

Keq for the reverse reaction

Cleavage & Debranching

1. Phosphorylase cleaves -1,4.

2. Transferase moves -1,4 to -1,4.

3. Glucosidase cleaves -1,6. This is a glucose not G-1-P

(Debranching enzyme)

These activities are in two enzymes in procaryotes and in a single multifunctional enzyme in eucaryotes.

Transferase and-1,6-Glucosidase

Proceeds through a diphospho intermediate.Similar to the phosphoglycerate mutase reaction.

Phosphoglucomutase

Glycogen PhosphorylaseThe enzyme is a dimer, PLP at each active site.

Pyridoxal Phosphate(PLP)

PLP is needed by phosphorylase for catalytic activity.

The phosphate is the site of conjugate acid/conjugate base activity.

Phosphorylase MechanismBoth glycogen and Glucose-1-P have bonds at the anomeric carbon so reaction can not be SN2.

Phosphorylase a & b

“a” has covalent P in “b” P is absent

Phosphorylase a(more active-has P) favors R state and phosphorylase b (less active-no P) favors T state.

Phosphorylase forms a & b,

Allosteric States T & R

Phosphorylase b (inactive, no P) is converted to phosphorylase a (active, has P) by the enzyme phosphorylase kinase (requires ATP).

Allosteric effectors of phosphorylase:AMP (+) b (favors R state)ATP (-) b (favors T state)Glucose-6-P (-) b (favors T state)Glucose (-) a (favors T state-liver)

P is removed from phosphorylase a by phosphoprotein phosphatase 1.

Phosphorylase a & b

Phosphorylase b

Phosphorylase a

Phosphorylase kinase

Phosphorylase kinase (PK), also called phosphorylase b kinase, is a large protein (1200000 daltons) with subunits (

The active site inis blocked unless P or Ca++ are presentis calmodulin, a small protein that binds Ca++.Ca++ increases the activity of PK.and are phosphorylation sites using protein kinase A and ATP. P increases the activity of PK.

Maximum activity requires both P and Ca++.

Phosphorylase kinase

Protein kinase A

Protein kinase A (cAMPdPK) is a tetramer of two regulatory subunits and two catalytic subunits.

R2C2 + cAMP -- > R2(cAMP) + 2 C

The unbound catalytic subunits are active and add P using ATP to both phosphorylase and glycogen synthase.

Protein kinase A is activated in response to hormonal binding outside the cell, G-protein transduction and activation of adenyl cyclase.

ATP -- > cAMP

Physiological Events

Glycogen breakdown

cascade

Amplification occurs at each step

Activation Cascade

Hormonal Activation

Epinephrine (adrenalin), a catechol amine, binds to receptors in muscle and liver to activate glycogenolysis. It also binds to receptors in liver to release Ca++. (See signal transduction, Ch 14.)

Regulation

This occurs at three levels depending upon needs of the cell/organism.

1. ATP/AMP ratio indicates normal energy need. ATP favors the inactive T state of phosphorylase b. AMP favors the active R state of phosphorylase b. ATP/AMP ratio does not affect phosphorylase a. Buildup of Glucose-6-P also favors the inactive T state of phosphorylase b.

2. Contracting muscle releases Ca++. Ca++ binds to calmodulin to partially activate PK.

Hormonal ActivationGlucagon, a 29 residue peptide, activates glycogenolysis in liver via the G-protein, Gs.

Regulation3. Hormonal stimulation.

Epinephrine binds to liver and muscle receptors to activate protein kinase A via the G-protein, Gs.In liver it also actives protein kinase C via the

G-protein, Gq and inositol phosphate route.

Glucagon binds only to liver receptors to activate protein kinase A via the G-protein, Gs.

Note: Regulation at phosphoprotein phosphatase 1 controls the reversal of these phosphorylation reactions.

Glycogen Synthesis

1. Glycogen synthesis may occur by adding glucose to existing glycogen structures. This requires:

1. hexokinase2. phosphoglucomutase3. UDP-glucose pyrophosphorylase4. glycogen synthase5. branching enzyme (glucosyl transferase)

2. Denovo synthesis starts a new glycogen molecule and requires the enzyme glycogenin in addition to those above. Glycogenin is a dimer, each subunit starts a short (1-4) strand on Tyr194 of the other subunit as a primer for glycogen synthase.

UDP Glucose

A high-energy glucose carrier

UDP-Glucose Synthesis

UDP-glucose pyrophosphorylase

G-1-P attacks P of UTP

Glycogen Synthesis

Glycogen Synthase

Adds only to the non-reducing ends of glycogen.

UTP is reformed using nucleosidediP kinase

ATP + UDP -- > ADP + UTP

Glycogen

This representationshows glycogenin on the reducing end of a glycogen molecule.

All of the non-reducing ends serve as substrate for breakdown or synthesis.

Synthase & Branching Enzyme

Glycogen synthase requires an -1,4 chain of at least 4 residues to be able to add another glucose. -1,4 addition continues until enough residues are present to permit branching.

Amylo-1,4 -- > 1,6-transglycosylase (glucosyl transferase) needs a strand of ~ 11 residues to act. It transfers ~6-7 residues to another strand to form a branch point and leaves at least 4 residues at the cleavage point.

Branching facilitates degradation and synthesis by providing substrate sites (non-reducing ends).

Denovo Synthesis

Glycogenin is a dimer of 37000 daltons per subunit. Each of the subunits catalyzes attachment of glucose to the other subunit. The first glucose is attached to Tyr 194 and requires UDPG and tyrosine glucosyl transferase activity. Then glycogenin adds ~7 more residues to form a short -1,4 chain again using UDPG as the glucose source. At this point glycogen synthase takes over along with the other enzymes noted previously.

Glycogenin remains permanently attached to the reducing end of the molecule.

Reciprocal RegulationGlycogenolysis initiated

Various kinases attack glycogen synthase at different sites, e.g. glycogen synthase kinase (GSK).

Protein Phosphatase 1 (PP1)

Loss of hormonal activation

Protein Phosphatase 1 (PP1)

PP1 is a 37000 dalton protein bound to several other proteins involved in regulation among which is the binding protein GM or GL. PP1 is active while bound to glycogen through GM.

When protein kinase A is active it forms PP1~P or GM~P which causes PP1 to dissociate from the binding protein GM. The unbound PP1 is less active.

Protein kinase A also activates protein phosphatase inhibitor (I~P) which then binds to the unbound PP1 to further decrease the activity of PP1.

Regulation of PP1

PP1 inactive

PP1 ContinuedUnder these conditions, PP1 activity is very, very low but not completely absent. When hormonal activation of the cell ceases the very low activity of PP1 slowly reverses the phosphorylation states of the system and glycogen synthesis can occur.

When insulin is bound to receptor, the effects of tyrosine kinase are seen. PP1 is phosphorylated at a site other than the one acted on by protein kinase A and PP1 activity is enhanced. The insulin stimulated pathway via TK also inactivates glycogen synthase kinase leading to glycogen synthesis.

InsulinInactivation of synthase

kinase

Insulin binding decreases blood glucose.

PP1 Continued

Liver does not have protein phosphatase inhibitor. Here, PP1 binds to phosphorylase a and is inactive while phosphorylase a is in the R state.

The number of phosphorylase molecules greatly out numbers those of PP1 resulting in negligible phosphatase activity while the R state is maintained.

As glucose levels rise, a shift occurs to the T state of phosphorylase a. PP1 then removes a Pi to produce phosphorylase b. These changes results in activation of PP1 as a result of dissociation of PP1 from phosphorylase.

Liver Phosphorylase & Glucose

End of Chapter 21

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition