Regulation of Glycogen Metabolism

• Protein Kinases

• Protein Phosphatases

• cAMP

• G proteins

• Calcitonin

• Insulin, glucagon, and epinephrine

Biochemical Definitions

1. Equilibrium: (Kinetics) When the rate of the forward reaction matches the rate of the reverse reaction. Does not specify quantity at equilibrium.

2. Equilibrium constant: (Thermodynamics) The quantity [B]/[A] at equilibrium. Does not specify rate.

4. Steady-state: A dynamic condition that allows flux without changing the concentration of components in the pathway.

3. Flux: Net carbon flow in one direction

A B CConsider

Connecting Kinetics with Thermodynamics

∆Go = RT ln Keq

Chemistry

Biochemistry

∆Go = RT ln Keq’

pH = 7.0 (10-7 M [H+] )

Practically Speaking…..

1. A negative ∆Go’ means [P] > [S] at equilbrium.

∆Go = RT ln Keq’

A B

2 -1.8 10 -5.9

100 -11.91,000 -17.8

10,000 -23.7100,000 -29.7

[B]/[A] ∆Go’ (kJ/mol)

Steady-State vs Equilibrium Reactions

Rule: An equilibrium reaction occurs in a closed system whereby two components, a reactant and a product, achieve a constancy of concentration based on chemical potentials

A B

Rule: A steady-state reaction occurs in an open system whereby two or more components achieve a constancy of concentration based on similar rates of components entering and leaving the system

A B ZY See Strategies p 163-167

See Strategies p 163-167

Ak1 k2

When k1 k2A is constant=

Representing a Steady-State

When k1 k2< A decreases

When k1 k2> A increases

Rule: A shift away from a dynamic steady-state evokes factors to restore the steady-state.

Rule: A shift away from a dynamic steady-state evokes factors to restore the steady-state.

Rule: Restoring steady-state requires modulating the activity of a rate-controlling enzyme(s) in the pathway

Rule: Restoring steady-state requires modulating the activity of a rate-controlling enzyme(s) in the pathway

Basics of Metabolic Homeostasis

1. Covalent modification

2. Changes in pathway [S] or enzyme cofactors3. Allosterism (Vmax or Km)

Enzyme activity can be modulated by:

4. Hormonal intervention

5. Enzyme turnover

Regulation of Carbon Flux• Net carbon flux through an individual step in a

pathway is defined as the difference between the forward and reverse reaction velocities

J = VF - VR (these are rates of change)

• At equilibrium: VF= VR; J = 0; i.e., there is no net flux even though VF and VR could be large

• When VF >> VR, J = VF

• At steady state, J = k (constant)

• At steady state, J depends on the rate determining (slowest) step in the pathway

• Net carbon flux through an individual step in a pathway is defined as the difference between the forward and reverse reaction velocities

J = VF - VR (these are rates of change)

• At equilibrium: VF= VR; J = 0; i.e., there is no net flux even though VF and VR could be large

• When VF >> VR, J = VF

• At steady state, J = k (constant)

• At steady state, J depends on the rate determining (slowest) step in the pathway

Textbook p591

A B C D Rate-determining

Lactate

Glucose

Rate-controlling Step

J = 0

VF > VR

Meaning of Flux

Flux varies with equilibrium position

1. If [A] = 100mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux would by 10%. (from 10:1 to 11:1)

2. If [A] = 20mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux by 33% . (2:1 to 3:1)

3. If [A] = 10mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux by 100% (1:1 to 2:1).

A B

A

A B

A

A B

A

AMP (0.1 mM)

Adenylate Kinase 2ADP = AMP + ATP

Rule: Regulators with the lowest steady-state concentration have the greatest impact on regulation

ATP (5 mM) ADP (1 mM)

ATP + X ATP + X~P + ADP5 0.5 4.5 0.5 0.5

2ADP ATP + AMP0.5 0.5 0.5

Final Talley: BeforeATP 5 mM

After5 mM No change

ADP 1 mM 1 mM No change

AMP 0.1 mM 0.6 mM 6 times

Why are there 2 enzymes in glycogen metabolism?

Glycogen

Glucose 1-PO4

Glycogenase

How can glycogensynthesis and degradationbe tuned to the needsof the cell? Only glycogenor glucose 1-PO4 concentrationcan affect VF or VR

Glycogen

Glucose 1-PO4UDP-Glucose

Phosphorylase Synthase

Because separate enzymescontrol synthesis and degradation, VF and VR

can vary depending on theenzyme that controls thedirection. This opens theway to allosteric and covalentcontrol of glycogen

Stimulating or inhibiting the enzyme cannot control direction

of flux

Stimulating or inhibiting the enzyme cannot control direction

of flux

cAMP Protein kinase (cAPK)cAMP Protein kinase (cAPK)

Ptase

Syn b

Syn a

ADP

PO4

ATP

Phos a

Phos bPtase Pkinase

PO4

ADP

ATP

Protein Kinase targets of cAPKProtein Kinase targets of cAPK

GlucagonEpinephrine

GlucagonEpinephrine

cAMPATP

Adenyl cyclase

Textbook p586

Pkinase

cAMP as a Regulator of Glycogen

cAMP Protein kinase (cAPK)cAMP Protein kinase (cAPK)

+ 4 cAMPC

CR R

CR

R

cAMP

cAMP

cAMP

cAMP

C+

C

2ATP 2ADP

P

P Active Kinase

Catalytic site

CalmodulinInactive KinasecAPK

Example would be phosphorylase b kinase

Example would be phosphorylase b kinase

Now its ready to phosphorylate

phosphorylase b

Now its ready to phosphorylate

phosphorylase b

Casein kinase II (primer)

Casein kinase II (primer)

P

Glycogen synthase kinase 3 (GSK3)

Glycogen synthase kinase 3 (GSK3)

PPP

Priming phosphateInactive

ATP

ADP

Glycogen Synthase

A

B

PP1PP1

H2O

3Pi

Inactivation of Glycogen Synthase

Phos a

Phos bPP1 Pkinase

PO4

ADP

ATP

PkinasePP1

Syn b

Syn a

ADP

PO4

ATP

Phosphoprotein Phosphatase-1 (PP1)Phosphoprotein Phosphatase-1 (PP1)

Phosphorylase Kinase a

Phosphorylase Kinase b

ADP

ATPPO4

H2O

PP1cAPK

Remember, this is the “stripper” enzyme. It takes “off” phosphate groups on proteins

Remember, this is the “stripper” enzyme. It takes “off” phosphate groups on proteins

Insulin stimulates glycogen synthesis in liver and muscle by blocking the action of GSK3 and activating PP1

GSK3

Glycogen Synthase a

Glycogen Synthase b

CKIIATP

3 ATP3 ATP

P

PP

Insulin

X

XInsulin

Glucagonepinephrine

Glucose-6-phosphate

Glucose

PP1ActiveInactive

3Pi

Text p586

inactive

Insulin

Insulin-stimulated protein kinase

Epinephrine, glucagon

cAPK

PP1Glycogen G subunit +

P

P

2ATP

2ADP

ADP

ATP

Glycogen synthesisstimulated,

breakdown blocked

Glycogen Breakdown stimulated,

synthesis blocked

more active

PP1Glycogen G subunit

P

OH

PP1Glycogen G subunit

OH

OH

ATP

ADP

(site 2 phosphorylation)

(site 1 and 2 phosphorylation)

(site 1 phosphorylation)

Phosphoprotein Phosphatase-1 (Muscle)

Less active

InhibitorP

K1

K2

K2

Text p588

Insulin regulation of GSK3

Text p 587

Shut down of GSK3 by PKB

Summary of Catabolic Hormonal Effects on Glycogen

• Glucagon and Epinephrine stimulate synthesis of cAMP• cAMP activates phosphorylase b kinase that converts

phosphorylase b to phosphorylase a• Phosphorylase a breaks down glycogen at an accelerated

rate• cAMP also inactivates PP1, prolonging the action of

phosphorylase a

Summary of Anabolic Effects on Glycogen

• Insulin activates glycogen synthase by stimulating phosphorylation of GDK3

• GDK3 inactivates itself allowing phosphoprotein phosphatase (PP1) to remove phosphate and activate glycogen synthase

• PPI also removes phosphate from phosphorylase a thereby shutting off glycogen breakdown

PAdenylcyclase

C C

R R

cAMP dependentprotein kinase

Phosphorylasekinase Phosphorylase

P

See Tutorial on cAMP-dependent Protein Kinases available on the web

Liver

PP

Glucose

T State R State

Phosphorylase a

Phosphorylase b

Active

Low GlucoseHigh Glucose

+5’-AMP

Binds weakly to b form

Phosphoprotein phosphatase-1

Low glucose a form (R state) is resistant to phosphatasePHigh glucose a form (T state) is vulnerable to phosphataseP

Text p446-447

Summary of Phosphoprotein Phosphatase-1 (Liver)

1. The Phosphatase binds to phosphorylase a (T or R form)

2. The T form has a serine exposed to allow hydrolysis of -PO4

3. The R form of phosphorylase a -PO4 cannot be hydrolyzed

4. Glucose converts R to T form which causes hydrolysis of -PO4 and dissociate the Phosphatase.

5. The liberated Phosphatase is free to activate glycogen synthase, thereby stimulating glycogen synthesis

(It does not bind to glycogen or a G protein)

(glucose is an allosteric effector of phosphorylase in liver)