biochemistry - i · biochemistry - i mondays and ... • ∆g’o, biochemical standard free...

Based on Profs. Kevin Gardner & Reza Khayat 1

Biochemistry - I

Mondays and Wednesdays 9:30-10:45 AM (MR-1307)

SPRING 2017

Lectures 17-18

Outline

•Metabolic regulation •Homeostasis •Achieving homeostasis •Points of regulation based on equilibrium •Coordinate regulation of glycolysis and gluconeogenesis •Glycogen synthesis •Regulation of glycogen catabolism via epinephrine/glucagon •Regulation of glycogen synthesis via insulin •Regulation of carbohydrate metabolism

Why learn about Principles of Metabolic Regulation? Every pathway we discuss is intricately intertwined with all other cellular pathways in a network of reactions. Therefore, any change in the reactants or products of a reaction not only affect that pathway but others as well. Problems or disruptions to these pathways give rise to many hereditary as well as acquired diseases.

3Chapter 15 - Metabolic Regulation: Glucose and Glycogen

Metabolic Regulation• 30,000 proteins, thousands of different reactions and

metabolites, and most shared by more than one pathway

• Enzymes responsible for pathways are under tight regulation by the cell via multiple mechanisms

• A lot of cross-talk between the different pathways

• Carbohydrates - a major energy source

• Stored as glycogen granules in vertebrates (right, hepatocyte) and starch in plants

• Glycogen in muscle provides quick source of energy

• More energy is stored in fats, but fats cannot be catabolized anaerobically, and take longer to catabolize

• http://www.genome.jp/kegg/pathway/map/map01100.html


Homeostasis

5

• Homeostasis: a property of a system that regulates its internal environment to maintain a stable and relatively constant condition of properties such as temperature, pH, and reaction flux

• Cells and organisms maintain a dynamic steady state • Dynamic because something is always happening. Many metabolic

pathways whose reactants and products are involved in other pathways • Steady because as fuel enters the cell (i.e. glucose) it is catabolized, and

the waste (i.e. CO2) product leaves the cell • The mass and gross composition of a typical cell does not change

appreciably over time (with the exception of aging and diseases) • Cells have mechanisms to regulate metabolic pathways to achieve homeostasis

- no backup of metabolites • Failure of mechanisms may be root of human diseases (homeostatic imbalance)

• diabetes • hypoglycemia • hyperglycemia • gout

• Conditions result from presence of increased particular metabolic product

Chapter 15 - Metabolic Regulation: Glucose and Glycogen

Factors Affecting Homeostasis


• Transcription - synthesis of mRNA from DNA • Transcriptome - set of genes that are being transcribed at any given point,

identified by next generation sequencing and gene chip/array methods.

• Translation - synthesis of protein from mRNA • Proteome - set of mRNAs being turned into proteins at any given point,

identified by mass spectrometry and protein chip/array methods.

• Reactants/products/activators/inhibitors - low molecular weight molecules, often generically called “metabolites”

• Metabolome - set of small molecule constituents of a cell at any given point, identified by mass spectrometry. Most importantly, this group addresses how the proteome can be regulated via proteins being reversibly turned “on” or “off” rather than degraded. For example, proteomic studies can indicate that the presence of a enzyme in the cell is identical in the diseased versus healthy state, but only metabolomics can identify that the enzyme, while being transcribed and translated equivalently in the two states, is more or less active

• Enzyme-catalyzed reactions can be modulated by: 1) the number of enzymes present, 2) catalytic activity of enzyme, and 3) concentration of their substrates and products

• Metabolic regulation: processes that hold cellular parameters (concentration of metabolites) constant over time even as the flow of metabolites changes

• Metabolic control: process that changes the output of a metabolic pathway over time

Achieving Homeostasis

(e.g. insulin, glucagon, etc.)

7

• Regulation of an enzymatic activity serves to regulate and control metabolism


Regulation Based on Equilibrium

8

• ∆G’o, biochemical standard free energy, occurs under a set of standard conditions (T=298K, P=1 atm, pH=7, all solutions = 1M) which are not the conditions in the cell!

• Keq intrinsic to chemical reaction; Q is controlled by cell via regulation of [E], [S], [effector/s]

• Reaction is near equilibrium when Q is < 2 orders of magnitude near Keq' • In multistep processes such as glycolysis, the cell maintains certain reactions (small Keq')

to be at steady state such that the direction of these reactions change with substrate concentration

• Concurrently, the cell maintains reactions with large Keq’ to be far from equilibrium, and thus as points of regulation (enzyme ON/OFF) for the overall pathway

15.1 Regulation of Metabolic Pathways 575

determines the magnitude and sign of !G" and thereforethe driving force, !G", of the reaction:

Because an alteration of this driving force profoundlyinfluences every reaction that involves ATP, organ-isms have evolved under strong pressure to developregulatory mechanisms responsive to the [ATP]/[ADP]ratio.

AMP concentration is an even more sensitive indi-cator of a cell’s energetic state than is [ATP]. Normally

¢G¿ # ¢G¿° $ RT ln [ADP][glucose 6-phosphate]

[ATP][glucose]

Equilibrium Constants, Mass Action Coefficients, and Free-Energy Changes for Enzymes of Carbohydrate Metabolism

Reactionnear !G"

Mass action ratio, Q equilibrium !G"! (kJ/mol)Enzyme K"eq Liver Heart in vivo?* (kJ/mol) in heart

Hexokinase 1 % 103 2 % 10&2 8 % 10&2 No &17 &27

PFK-1 1.0 % 103 9 % 10&2 3 % 10&2 No &14 &23

Aldolase 1.0 % 10&4 1.2 % 10&6 9 % 10&6 Yes $24 &6.0

Triose phosphate isomerase 4 % 10&2 — 2.4 % 10&1 Yes $7.5 $3.8

Glyceraldehyde 3-phosphatedehydrogenase $phosphoglycerate kinase 2 % 103 6 % 102 9.0 Yes &13 $3.5

Phosphoglycerate mutase 1 % 10&1 1 % 10&1 1.2 % 10&1 Yes $4.4 $0.6

Enolase 3 2.9 1.4 Yes &3.2 &0.5

Pyruvate kinase 2 % 104 7 % 10&1 40 No &31 &17

Phosphoglucose isomerase 4 % 10&1 3.1 % 10&1 2.4 % 10&1 Yes $2.2 &1.4

Pyruvate carboxylase $ PEP carboxykinase 7 1 % 10&3 — No &5.0 &23

Glucose 6-phosphatase 8.5 % 102 1.2 % 102 — Yes &17 &5.0

TABLE 15–3

Source: K"eq and Q from Newsholme, E.A. & Start, C. (1973) Regulation in Metabolism, Wiley Press, New York, pp. 97, 263. !G" and !G"' were calculated from these data.

*For simplicity, any reaction for which the absolute value of the calculated !G" is less than 6 is considered near equilibrium.

Initi

al v

eloc

ity, V

(arb

itrar

y un

its)

ATP concentration [mM]

5 10 15 20 25 30 35 40

Vmax12

Vmax

FIGURE 15–5 Effect of ATP concentration on the initial velocity of atypical ATP-dependent enzyme. These experimental data yield a Km forATP of 5 mM. The concentration of ATP in animal tissues is !5 mM.

Adenine Nucleotides Play Special Roles in Metabolic Regulation

After the protection of its DNA from damage, perhapsnothing is more important to a cell than maintaining aconstant supply and concentration of ATP. Many ATP-using enzymes have Km values between 0.1 and 1 mM,and the ATP concentration in a typical cell is about 5 mM.If [ATP] were to drop significantly, these enzymes wouldbe less than fully saturated by their substrate (ATP), andthe rates of hundreds of reactions that involve ATPwould decrease (Fig. 15–5); the cell would probably notsurvive this kinetic effect on so many reactions.

There is also an important thermodynamic effectof lowered [ATP]. Because ATP is converted to ADPor AMP when “spent” to accomplish cellular work,the [ATP]/[ADP] ratio profoundly affects all reactionsthat employ these cofactors. (The same is true forother important cofactors, such as NADH/NAD$ andNADPH/NADP$.) For example, consider the reactioncatalyzed by hexokinase:

Note that this expression holds true only when reac-tants and products are at their equilibrium concentra-tions, where !G" # 0. At any other set of concentrations,!G" is not zero. Recall (from Chapter 13) that the ratioof products to substrates (the mass action ratio, Q)

K¿eq #[ADP]eq[glucose 6-phosphate]eq

[ATP]eq[glucose]eq# 2 % 103

ATP $ glucose ¡ ADP $ glucose 6-phosphate

13

7

10

‘

Bioenergetics and Biochemical Reaction Types492

KEY CONVENTION: In another simplifying convention usedby biochemists, when H2O, H!, and/or Mg2! are reac-tants or products, their concentrations are not includedin equations such as Equation 13–2 but are insteadincorporated into the constants K"eq and #G"$. ■

Just as K"eq is a physical constant characteristic foreach reaction, so too is #G"$ a constant. As we noted inChapter 6, there is a simple relationship between K"eq

and #G"$:

(13–3)

The standard free-energy change of a chemical reac-tion is simply an alternative mathematical way ofexpressing its equilibrium constant. Table 13–2shows the relationship between #G"$ and K"eq. If theequilibrium constant for a given chemical reaction is 1.0,the standard free-energy change of that reaction is 0.0(the natural logarithm of 1.0 is zero). If K"eq of a reactionis greater than 1.0, its #G"$ is negative. If K"eq is less than1.0, #G"$ is positive. Because the relationship between#G"$ and K"eq is exponential, relatively small changes in#G"$ correspond to large changes in K"eq.

It may be helpful to think of the standard free-energy change in another way. #G"$ is the difference be-tween the free-energy content of the products and thefree-energy content of the reactants, under standardconditions. When #G"$ is negative, the products containless free energy than the reactants and the reaction willproceed spontaneously under standard conditions; allchemical reactions tend to go in the direction that re-sults in a decrease in the free energy of the system. A

¢G¿° % &RT ln K¿eq

positive value of #G"$ means that the products of the re-action contain more free energy than the reactants, andthis reaction will tend to go in the reverse direction if westart with 1.0 M concentrations of all components (stan-dard conditions). Table 13–3 summarizes these points.

Table 13–4 gives the standard free-energy changesfor some representative chemical reactions. Note thathydrolysis of simple esters, amides, peptides, and glyco-sides, as well as rearrangements and eliminations, pro-ceed with relatively small standard free-energy changes,whereas hydrolysis of acid anhydrides is accompaniedby relatively large decreases in standard free energy.The complete oxidation of organic compounds such asglucose or palmitate to CO2 and H2O, which in cells re-quires many steps, results in very large decreases instandard free energy. However, standard free-energy

WORKED EXAMPLE 13–1 Calculation of !G"#

Calculate the standard free-energy change of the reac-tion catalyzed by the enzyme phosphoglucomutase

Glucose 1-phosphate ∆ glucose 6-phosphate

given that, starting with 20 mM glucose 1-phosphate andno glucose 6-phosphate, the final equilibrium mixture at25 $C and pH 7.0 contains 1.0 mM glucose 1-phosphateand 19 mM glucose 6-phosphate. Does the reaction inthe direction of glucose 6-phosphate formation proceedwith a loss or a gain of free energy?

Solution: First we calculate the equilibrium constant:

We can now calculate the standard free-energy change:

Because the standard free-energy change is negative,the conversion of glucose 1-phosphate to glucose 6-phosphate proceeds with a loss (release) of freeenergy. (For the reverse reaction, #G"$ has the samemagnitude but the opposite sign.)

% &7.3 kJ/mol % &(8.315 J/mol $K)(298 K)(ln 19)

¢G¿° % &RT ln K¿eq

K¿eq %[glucose 6-phosphate][glucose 1-phosphate]

%19 mM

1.0 mM% 19

Relationship between EquilibriumConstants and Standard Free-EnergyChanges of Chemical Reactions

TABLE 13–2

#G"#

K"eq (kJ/mol) (kcal/mol)*

103 &17.1 &4.1

102 &11.4 &2.7

101 &5.7 &1.4

1 0.0 0.0

10&1 5.7 1.4

10&2 11.4 2.7

10&3 17.1 4.1

10&4 22.8 5.5

10&5 28.5 6.8

10&6 34.2 8.2

*Although joules and kilojoules are the standard units of energy and are used through-out this text, biochemists and nutritionists sometimes express #G"$ values in kilocalo-ries per mole. We have therefore included values in both kilojoules and kilocalories inthis table and in Tables 13–4 and 13–6. To convert kilojoules to kilocalories, divide thenumber of kilojoules by 4.184.

TABLE 13–3

Starting with allcomponents at 1 M,

When K"eq is . . . #G"# is . . . the reaction . . .

'1.0 negative proceeds forward

1.0 zero is at equilibrium

(1.0 positive proceeds in reverse

Relationships among K"eq, #G"#, and the Direction of Chemical Reactions

13.1 Bioenergetics and Thermodynamics 493

changes such as those in Table 13–4 indicate how muchfree energy is available from a reaction under standardconditions. To describe the energy released under theconditions existing in cells, an expression for the actualfree-energy change is essential.

Actual Free-Energy Changes Depend on Reactant and Product Concentrations

We must be careful to distinguish between two differentquantities: the actual free-energy change, !G, and thestandard free-energy change, !G"#. Each chemical reac-tion has a characteristic standard free-energy change,which may be positive, negative, or zero, depending onthe equilibrium constant of the reaction. The standardfree-energy change tells us in which direction and howfar a given reaction must go to reach equilibrium whenthe initial concentration of each component is 1.0 M,the pH is 7.0, the temperature is 25 #C, and the pressureis 101.3 kPa (1 atm). Thus !G"# is a constant: it has acharacteristic, unchanging value for a given reaction.But the actual free-energy change, !G, is a function ofreactant and product concentrations and of the temper-ature prevailing during the reaction, none of which will

necessarily match the standard conditions as definedabove. Moreover, the !G of any reaction proceedingspontaneously toward its equilibrium is always negative,becomes less negative as the reaction proceeds, and iszero at the point of equilibrium, indicating that no morework can be done by the reaction.

!G and !G"# for any reaction aA $ bB ∆ cC $ dDare related by the equation

(13–4)

in which the terms in red are those actually prevailingin the system under observation. The concentrationterms in this equation express the effects commonlycalled mass action, and the term [C]c[D]d/[A]a[B]b is calledthe mass-action ratio, Q. Thus Equation 13–4 can beexpressed as !G % !G"# $ RT ln Q. As an example, let ussuppose that the reaction A $ B ∆ C $ D is taking placeunder the standard conditions of temperature (25 #C) andpressure (101.3 kPa) but that the concentrations of A, B, C, and D are not equal and none of the compo-nents is present at the standard concentration of 1.0 M. To determine the actual free-energy change, !G, underthese nonstandard conditions of concentration as the

¢G % ¢G¿° $ RT ln [C]c[D]d

[A]a[B]b

Standard Free-Energy Changes of Some Chemical ReactionsTABLE 13–4

!G!"

Reaction type (kJ/mol) (kcal/mol)

Hydrolysis reactions

Acid anhydrides

Acetic anhydride $ H2O 88n 2 acetate &91.1 &21.8ATP $ H2O 88n ADP $ Pi &30.5 &7.3ATP $ H2O 88n AMP $ PPi &45.6 &10.9PPi $ H2O 88n 2Pi &19.2 &4.6UDP-glucose $ H2O 88n UMP $ glucose 1-phosphate &43.0 &10.3

Esters

Ethyl acetate $ H2O 88n ethanol $ acetate &19.6 &4.7Glucose 6-phosphate $ H2O 88n glucose $ Pi &13.8 &3.3

Amides and peptides

Glutamine $ H2O 88n glutamate $ NH$4 &14.2 &3.4

Glycylglycine $ H2O 88n 2 glycine &9.2 &2.2

Glycosides

Maltose $ H2O 88n 2 glucose &15.5 &3.7Lactose $ H2O 88n glucose $ galactose &15.9 &3.8

Rearrangements

Glucose 1-phosphate 88n glucose 6-phosphate &7.3 &1.7Fructose 6-phosphate 88n glucose 6-phosphate &1.7 &0.4

Elimination of water

Malate 88n fumarate $ H2O 3.1 0.8

Oxidations with molecular oxygen

Glucose $ 6O2 88n 6CO2 $ 6H2O &2,840 &686Palmitate $ 23O2 88n 16CO2 $ 16H2O &9,770 &2,338

13.1 Bioenergetics and Thermodynamics 493

changes such as those in Table 13–4 indicate how muchfree energy is available from a reaction under standardconditions. To describe the energy released under theconditions existing in cells, an expression for the actualfree-energy change is essential.

Actual Free-Energy Changes Depend on Reactant and Product Concentrations

We must be careful to distinguish between two differentquantities: the actual free-energy change, !G, and thestandard free-energy change, !G"#. Each chemical reac-tion has a characteristic standard free-energy change,which may be positive, negative, or zero, depending onthe equilibrium constant of the reaction. The standardfree-energy change tells us in which direction and howfar a given reaction must go to reach equilibrium whenthe initial concentration of each component is 1.0 M,the pH is 7.0, the temperature is 25 #C, and the pressureis 101.3 kPa (1 atm). Thus !G"# is a constant: it has acharacteristic, unchanging value for a given reaction.But the actual free-energy change, !G, is a function ofreactant and product concentrations and of the temper-ature prevailing during the reaction, none of which will

necessarily match the standard conditions as definedabove. Moreover, the !G of any reaction proceedingspontaneously toward its equilibrium is always negative,becomes less negative as the reaction proceeds, and iszero at the point of equilibrium, indicating that no morework can be done by the reaction.

!G and !G"# for any reaction aA $ bB ∆ cC $ dDare related by the equation

(13–4)

in which the terms in red are those actually prevailingin the system under observation. The concentrationterms in this equation express the effects commonlycalled mass action, and the term [C]c[D]d/[A]a[B]b is calledthe mass-action ratio, Q. Thus Equation 13–4 can beexpressed as !G % !G"# $ RT ln Q. As an example, let ussuppose that the reaction A $ B ∆ C $ D is taking placeunder the standard conditions of temperature (25 #C) andpressure (101.3 kPa) but that the concentrations of A, B, C, and D are not equal and none of the compo-nents is present at the standard concentration of 1.0 M. To determine the actual free-energy change, !G, underthese nonstandard conditions of concentration as the

¢G % ¢G¿° $ RT ln [C]c[D]d

[A]a[B]b

Standard Free-Energy Changes of Some Chemical ReactionsTABLE 13–4

!G!"

Reaction type (kJ/mol) (kcal/mol)

Hydrolysis reactions

Acid anhydrides

Acetic anhydride $ H2O 88n 2 acetate &91.1 &21.8ATP $ H2O 88n ADP $ Pi &30.5 &7.3ATP $ H2O 88n AMP $ PPi &45.6 &10.9PPi $ H2O 88n 2Pi &19.2 &4.6UDP-glucose $ H2O 88n UMP $ glucose 1-phosphate &43.0 &10.3

Esters

Ethyl acetate $ H2O 88n ethanol $ acetate &19.6 &4.7Glucose 6-phosphate $ H2O 88n glucose $ Pi &13.8 &3.3

Amides and peptides

Glutamine $ H2O 88n glutamate $ NH$4 &14.2 &3.4

Glycylglycine $ H2O 88n 2 glycine &9.2 &2.2

Glycosides

Maltose $ H2O 88n 2 glucose &15.5 &3.7Lactose $ H2O 88n glucose $ galactose &15.9 &3.8

Rearrangements

Glucose 1-phosphate 88n glucose 6-phosphate &7.3 &1.7Fructose 6-phosphate 88n glucose 6-phosphate &1.7 &0.4

Elimination of water

Malate 88n fumarate $ H2O 3.1 0.8

Oxidations with molecular oxygen

Glucose $ 6O2 88n 6CO2 $ 6H2O &2,840 &686Palmitate $ 23O2 88n 16CO2 $ 16H2O &9,770 &2,338

Q

Two Key Hormones with Opposing Functions

9

Glucagon: A peptide hormone released by the pancreas to signal low [glucose] in the blood, and tells the liver to release glucose into the blood from glycogen or gluconeogenesis

Insulin: A peptide hormone released by pancreas to signal a high [glucose] in the blood, and tells the liver to absorb glucose for storage as glycogen, glycolysis…


• Reciprocal regulation of glycolysis and gluconeogenesis • Glycogen breakdown • Glycogen synthesis • Regulation of glycogen breakdown • Regulation of glycogen synthesis • Regulation of carbohydrate metabolism

Topics


Coordinated Regulation of Glycolysis and Gluconeogenesis

• The liver has the special role of maintaining a constant [glucose] • Low blood [glucose] results in glucagon being released to signal liver to stop glucose

consumption and increase glucose release

• Isozymes • Evolved from a common gene (paralogs) • Encoded by different genes • May occur in the same cell • May differ in their use of cofactors, kinetic

parameters, and intracellular distribution (e.g. cytosolic vs. membrane)

Regulation involves enzymes bypassed for glycolysis/gluconeogenesis:

Hexokinase Phosphofructokinase-1 (PFK-1) Pyruvate kinase


Regulating Glycolytic Flux via Hexokinase (R1, glycolysis)

• Hexokinase has four different isozymes (I-IV) • Muscle isozymes I, II, and III are inhibited by

their product (glucose 6-P) • Hexokinase IV (hepatocyte/liver) has a lower

affinity (= higher Km) for glucose and not inhibited by its product (glucose 6-P)

• An efficient glucose transporter (GLUT2) equilibrates glucose in liver and blood. Thus, low blood [glucose] = low hepatocyte/liver [glucose]

• Isozyme IV activity decreases as [glucose] decreases

• Glycolysis slows down, and glucose is released into blood via GLUT2


Regulation of Hexokinase IV (R1, Glycolysis)• Hexokinase IV inhibited by a regulatory protein specific to hepatocytes (liver) • Regulatory protein binds fructose 6-phosphate (F6P) and glucose

• Glycolysis produces F6P (R2) • At high [F6P], regulatory protein binds hexokinase IV and recruits it to nucleus where

its substrate is not present; thus no G6P produced • At high [glucose], regulatory protein releases hexokinase IV and allows it to return to

cytosol to become active • Glucose and F6P compete to activate/deactivate hexokinase IV • Hexokinase IV and glucose-6-phosphatase are reciprocally regulated at the level of

transcription


Regulation of Phosphofructokinase-1 (R3, Glycolysis)

ATP substrate

allosteric regulator

(two of four subunits shown)

• PFK-1 is the “committed” step to glycolysis, and under allosteric regulation (graph on the right)

• Has a catalytic and allosteric site (bottom right). ADP in blue and fructose 1,6-bisphophate in yellow

• Inhibited allosterically by ATP • high [ATP] lowers affinity of enzyme for its substrate • high [ATP] binds to allosteric binding site


Reciprocal Regulation of Glycolysis (R3) & Gluconeogenesis via PFK-1

• AMP, the allosteric activator of PFK-1, is an allosteric inhibitor of FBPase-1

• High [citrate] indicates that cells are meeting energy needs via TCA

• Gluconeogenesis favored at high [acetyl-CoA], [citrate] , or [ATP]


F2,6BP

• F2,6BP increases PFK-1 affinity for substrate and reduces its affinity for inhibitors (ATP and citrate); hence it stimulates glycolysis in liver

• F2,6BP concurrently inhibits FBPase-1; hence it inhibits gluconeogenesis in liver • F2,6BP is a regulatory molecule, not an intermediate in the pathway


Hormonal Regulation of Glycolysis & Gluconeogenesis

Regulation of Pyruvate Kinase (R10, glycolysis)

• 3 isozymes differ in tissue distribution • All inhibited by high concentrations of ATP, acetyl CoA, and long chain fatty acids (all

signs of abundant energy) • Activated by F1,6BP • Glucagon is released into blood at low [glucose], activates cAMP dependent protein

kinase A (PKA) which phosphorylates and inactivates the L isozyme (below) • This slows the use of glucose for fuel in liver, sparing it for brain and other organs

17

(PP = Protein phosphatase)

15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 589

their response to modulators. High concentrations ofATP, acetyl-CoA, and long-chain fatty acids (signsof abundant energy supply) allosterically inhibit allisozymes of pyruvate kinase (Fig. 15–19). The liverisozyme (L form), but not the muscle isozyme (Mform), is subject to further regulation by phosphoryla-tion. When low blood glucose causes glucagon release,cAMP-dependent protein kinase phosphorylates the Lisozyme of pyruvate kinase, inactivating it. This slowsthe use of glucose as a fuel in liver, sparing it for exportto the brain and other organs. In muscle, the effect ofincreased [cAMP] is quite different. In response to epi-nephrine, cAMP activates glycogen breakdown andglycolysis and provides the fuel needed for the fight-or-flight response.

Catalyticsubunit

RegulatorysubunitInhibitor

2 Mn2+

Scaffold/A subunit

(a) (b)Scaffold/

A subunitCatalyticsubunit

Regulatorysubunit 1

Regulatorysubunit 2

Substrate-recognitionsurface 2

Substrate-recognitionsurface 1

Holoenzyme 1 Holoenzyme 2

FIGURE 15–18 Structure and action of phosphoprotein phosphatase2A (PP2A). (a) The catalytic subunit has two Mn2! ions in its activesite, positioned close to the substrate recognition surface formed by theinterface between the catalytic subunit and the regulatory subunit(PDB ID 2NPP). Microcystin-LR, shown here in red, is a specific in-hibitor of PP2A. The catalytic and regulatory subunits rest in a scaffold(the A subunit) that positions them relative to each other and shapesthe substrate recognition site. (b) PP2A recognizes several target pro-teins, its specificity provided by the regulatory subunit. Each of severalregulatory subunits fits the scaffold containing the catalytic subunit,and each regulatory subunit creates its unique substrate-binding site.

Liver only All glycolytic tissues, including liver

glucagon

ADP

ADP

ATP

ATP

ATP,acetyl-CoA,long-chain fatty acids

PKA

P

Pyruvatekinase

L/M

Pyruvatekinase L(inactive)

H2OPP

Pi

PEP

Pyruvate

transamination

Alanine

F16BP

6 steps

FIGURE 15–19 Regulation of pyruvate kinase. The enzyme is allosteri-cally inhibited by ATP, acetyl-CoA, and long-chain fatty acids (all signsof an abundant energy supply), and the accumulation of fructose 1,6-bisphosphate triggers its activation. Accumulation of alanine, whichcan be synthesized from pyruvate in one step, allosterically inhibitspyruvate kinase, slowing the production of pyruvate by glycolysis. Theliver isozyme (L form) is also regulated hormonally. Glucagon activates

cAMP-dependent protein kinase (PKA; see Fig. 15–35), which phos-phorylates the pyruvate kinase L isozyme, inactivating it. When theglucagon level drops, a protein phosphatase (PP) dephosphorylatespyruvate kinase, activating it. This mechanism prevents the liver fromconsuming glucose by glycolysis when blood glucose is low; instead,the liver exports glucose. The muscle isozyme (M form) is not affectedby this phosphorylation mechanism.


Regulating the Fate of Pyruvate

18

Principles of Metabolic Regulation590

Energy

Citric acid cycle

Oxaloacetate

pyruvatecarboxylase

Gluconeogenesis

Glucose

Pyruvatepyruvate

dehydrogenasecomplex

Acetyl-CoA

CO2

FIGURE 15–20 Two alternative fates for pyruvate. Pyruvate can beconverted to glucose and glycogen via gluconeogenesis or oxidized toacetyl-CoA for energy production. The first enzyme in each path is reg-ulated allosterically; acetyl-CoA, produced either by fatty acid oxida-tion or by the pyruvate dehydrogenase complex, stimulates pyruvatecarboxylase and inhibits pyruvate dehydrogenase.

The Gluconeogenic Conversion of Pyruvate to PhosphoenolPyruvate Is Under Multiple Types of Regulation

In the pathway leading from pyruvate to glucose, thefirst control point determines the fate of pyruvate in themitochondrion: its conversion either to acetyl-CoA (bythe pyruvate dehydrogenase complex) to fuel the citricacid cycle (Chapter 16) or to oxaloacetate (by pyruvatecarboxylase) to start the process of gluconeogenesis(Fig. 15–20). When fatty acids are readily available asfuels, their breakdown in liver mitochondria yieldsacetyl-CoA, a signal that further oxidation of glucose forfuel is not necessary. Acetyl-CoA is a positive allostericmodulator of pyruvate carboxylase and a negative mod-ulator of pyruvate dehydrogenase, through stimulationof a protein kinase that inactivates the dehydrogenase.When the cell’s energy needs are being met, oxidativephosphorylation slows, NADH rises relative to NAD!

and inhibits the citric acid cycle, and acetyl-CoA accu-mulates. The increased concentration of acetyl-CoA in-hibits the pyruvate dehydrogenase complex, slowing theformation of acetyl-CoA from pyruvate, and stimulatesgluconeogenesis by activating pyruvate carboxylase,

allowing conversion of excess pyruvate to oxaloacetate(and, eventually, glucose).

Oxaloacetate formed in this way is converted tophosphoenolpyruvate (PEP) in the reaction catalyzedby PEP carboxykinase (Fig. 15–11). In mammals, theregulation of this key enzyme occurs primarily at thelevel of its synthesis and breakdown, in response to di-etary and hormonal signals. Fasting or high glucagonlevels act through cAMP to increase the rate of tran-scription and to stabilize the mRNA. Insulin, or highblood glucose, has the opposite effects. We discuss thistranscriptional regulation in more detail below. Gener-ally triggered by a signal from outside the cell (diet, hor-mones), these changes take place on a time scale ofminutes to hours.

Transcriptional Regulation of Glycolysis andGluconeogenesis Changes the Number of Enzyme Molecules

Most of the regulatory actions discussed thus far aremediated by fast, quickly reversible mechanisms: al-losteric effects, covalent alteration (phosphorylation)of the enzyme, or binding of a regulatory protein. An-other set of regulatory processes involves changes inthe number of molecules of an enzyme in the cell,through changes in the balance of enzyme synthesisand breakdown, and our discussion now turns to regu-lation of transcription through signal-activated tran-scription factors.

In Chapter 12 we encountered nuclear receptorsand transcription factors in the context of insulin signal-ing. Insulin acts through its receptor in the plasma mem-brane to turn on at least two distinct signaling pathways,each involving activation of a protein kinase. The MAPkinase ERK, for example, phosphorylates the transcrip-tion factors SRF and Elk1 (see Fig. 12–15), which thenstimulate the synthesis of enzymes needed for cellgrowth and division. Protein kinase B (PKB; also calledAkt) phosphorylates another set of transcription factors(PDX1, for example), and these stimulate the synthesisof enzymes that metabolize carbohydrates and the fatsformed and stored following excess carbohydrate intakein the diet. In pancreatic ! cells, PDX1 also stimulatesthe synthesis of insulin itself.

More than 150 genes are transcriptionally regulatedby insulin; humans have at least seven general types ofinsulin response elements, each recognized by a subsetof transcription factors activated by insulin under vari-ous conditions. Insulin stimulates the transcription of thegenes that encode hexokinases II and IV, PFK-1, pyru-vate kinase, and PFK-2/FBPase-2 (all involved in glycol-ysis and its regulation); several enzymes of fatty acidsynthesis; and glucose 6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase, enzymes of thepentose phosphate pathway that generate the NADPHrequired for fatty acid synthesis. Insulin also slows the

Gluconeogenesis

Oxidative phosphorylation

occurs in mitochondrion

occurs in mitochondrion



Topics


Glycogen Breakdown, a Source of Glucose

• Glycogen breakdown is catalyzed by glycogen phosphorylase (first enzyme)

• Occurs predominantly in liver (10% mass) and little in muscle (1-2% mass)

• Glycogen phosphorylase removes terminal glucose from nonreducing end of glycogen

• Repetitive removal of glucose monomers (G1-P) until it reaches the fourth glucose from a branch point

• Then a “debranching” and bifunctional enzyme uses its transferase activity through two successive reactions to debranch and transfer branches

• First, the (α1-4) bonds are cleaved to remove three residue oligosaccharide

• transfer oligosaccharide to neighboring chain • the (α1-6) bonds cleaved


Glycogen Breakdown• G1-P, the end product of the glycogen phosphorylase reaction can enter

glycolysis in liver, or replenish blood glucose • Phosphoglucomutase (below) donates a PO4 (from phosphoserine) to glucose 1-

P, then the P at C1 is transferred to the enzyme

21


bond is preserved in the formation of the phosphate ester,glucose 1-phosphate (see Section 14.2).

Pyridoxal phosphate is an essential cofactor in theglycogen phosphorylase reaction; its phosphate groupacts as a general acid catalyst, promoting attack by Pi onthe glycosidic bond. (This is an unusual role for pyri-doxal phosphate; its more typical role is as a cofactor inamino acid metabolism; see Fig. 18–6.)

Glycogen phosphorylase acts repetitively on thenonreducing ends of glycogen branches until it reachesa point four glucose residues away from an (!1n6)branch point (see Fig. 7–14), where its action stops.Further degradation by glycogen phosphorylase can oc-cur only after the debranching enzyme, formallyknown as oligo (!1n6) to (!1n4) glucan-trans-ferase, catalyzes two successive reactions that transferbranches (Fig. 15–26). Once these branches are trans-ferred and the glucosyl residue at C-6 is hydrolyzed,glycogen phosphorylase activity can continue.

Glucose 1-Phosphate Can Enter Glycolysis or, in Liver,Replenish Blood Glucose

Glucose 1-phosphate, the end product of the glycogenphosphorylase reaction, is converted to glucose 6-phos-phate by phosphoglucomutase, which catalyzes thereversible reaction

Initially phosphorylated at a Ser residue, the enzyme do-nates a phosphoryl group to C-6 of the substrate, thenaccepts a phosphoryl group from C-1 (Fig. 15–27).

The glucose 6-phosphate formed from glycogen inskeletal muscle can enter glycolysis and serve as an en-ergy source to support muscle contraction. In liver,glycogen breakdown serves a different purpose: to re-lease glucose into the blood when the blood glucoselevel drops, as it does between meals. This requires theenzyme glucose 6-phosphatase, present in liver and kid-ney but not in other tissues. The enzyme is an integralmembrane protein of the endoplasmic reticulum, pre-dicted to contain nine transmembrane helices, with itsactive site on the lumenal side of the ER. Glucose 6-phosphate formed in the cytosol is transported into theER lumen by a specific transporter (T1) (Fig. 15–28)and hydrolyzed at the lumenal surface by the glucose 6-phosphatase. The resulting Pi and glucose are thoughtto be carried back into the cytosol by two differenttransporters (T2 and T3), and the glucose leaves the he-patocyte via the plasma membrane transporter, GLUT2.Notice that by having the active site of glucose 6-phos-phatase inside the ER lumen, the cell separates this re-action from the process of glycolysis, which takes placein the cytosol and would be aborted by the action ofglucose 6-phosphatase. Genetic defects in either glu-cose 6-phosphatase or T1 lead to serious derangementof glycogen metabolism, resulting in type Ia glycogenstorage disease (Box 15–4).

Glucose 1-phosphate ∆ glucose 6-phosphate

Because muscle and adipose tissue lack glucose 6-phosphatase, they cannot convert the glucose 6-phos-phate formed by glycogen breakdown to glucose, andthese tissues therefore do not contribute glucose to theblood.

The Sugar Nucleotide UDP-Glucose Donates Glucose for Glycogen Synthesis

Many of the reactions in which hexoses are transformedor polymerized involve sugar nucleotides, compoundsin which the anomeric carbon of a sugar is activated by attachment to a nucleotide through a phosphate esterlinkage. Sugar nucleotides are the substrates for polymer-ization of monosaccharides into disaccharides, glycogen,

Glucose 1-phosphatemolecules

(a1→ 6)glucosidase

activity ofdebranching

enzyme

transferaseactivity of

debranchingenzyme

Unbranched (a1→ 4) polymer;substrate for furtherphosphorylase action

glycogenphosphorylase

(a1→ 6)linkage

Nonreducingends

Glycogen

Glucose

FIGURE 15–26 Glycogen breakdown near an (!1→6) branch point.Following sequential removal of terminal glucose residues by glycogenphosphorylase (see Fig. 15–25), glucose residues near a branch are re-moved in a two-step process that requires a bifunctional debranchingenzyme. First, the transferase activity of the enzyme shifts a block ofthree glucose residues from the branch to a nearby nonreducing end,to which they are reattached in (!1n4) linkage. The single glucoseresidue remaining at the branch point, in (!1n6) linkage, is then re-leased as free glucose by the debranching enzyme’s (!1n6) glucosi-dase activity. The glucose residues are shown in shorthand form, whichomits the ⎯H, ⎯OH, and ⎯CH2OH groups from the pyranose rings.

• Glycogen catabolism in muscle serves as an energy source to support muscle contraction

• Glycogen catabolism in liver serves to release glucose into blood for other organs


Glucose-6P Transported Out of the Cell

22

• In the liver, glucose 6-P is transported from cytoplasm into Endoplasmic reticulum via the G6P transporter (T1) down a chemical gradient

• G6P is dephosphorylated by glucose 6-Pase (present only in liver), and pumped out of ER by a glucose transporter down a chemical gradient

• Glucose is transported out of cytoplasm, and into blood, via GLUT2 down a chemical gradient

• G6P sequestration outcompetes/inhibits glycolysis



Topics


Glycogen Synthesis• Occurs in all tissues, but predominant in liver • The sugar nucleotide UDP-glucose donates

glucose for glycogen synthesis

• Many reactions which polymerize hexoses involve sugar nucleotides formed through a condensation reaction between a nucleoside triphosphate and a sugar phosphate

• Hydrolysis of PPi to 2Pi pulls reaction forward by removing the product of the reaction

• Nucleotide sugars are: • Stable (formation irreversible) • Nucleotide allows more contact points

with enzymes • “Tagging” the sugars with nucleotidyl

groups sets aside a pool dedicated to one purpose

• Nucleotides are excellent leaving groups


Glycogen Synthesis

• Glycogenin initiates the synthesis of glycogen

• Transfers a glucose from UDP-glucose to its own Tyr

• Adds 7 more glucose residues from UDP-glucose (total of 8 glucose)

• Glycogen synthase takes over


• Glycogen synthase transfers a glucose residue to the nonreducing end of existing glycogen branch, and continues to makes new α1-4 linkage for ~11 glucose molecules

• Amylo transglycolase (branching enzyme) moves 6 or 7 glucose residues from 11 glucose chain to form an α1-6 linkage with the first glucose

26

12311 45678910

1234

56711 8910


Glycogen synthesis, continued

• Glycogenin remains buried within the particle

• 12 tiers of glucose chains in glycogen particle (only 5 shown here).

• Total of about 55,000 glucose residues (21 nm diameter, MW ~ 107)

• Inner chains have two α1-6 linkages each, chains in outer tier are unbranched

• Many nonreducing ends allow for rapid glycogen catabolism

27

1

2

3

4

5


Glycogen Synthesis


Topics


Regulation of Glycogen Catabolism via Ephinephrine & Glucagon

• Hormones epinephrine and glucagon initiate “cascade” mechanisms that regulate cellular activity

• In muscle: provides glucose for ATP via glycolysis • In liver: provides glucose for blood

• Both trigger generation of cAMP

• cAMP activates PKA (protein kinase A)

• PKA activates phosphorylase b kinase

• Phosphorylase b kinase activates glycogen phosphorylase

• Two interconvertible forms of glycogen phosphorylase • glycogen phosphorylase a (active) • glycogen phosphorylase b (less active, default)

• The glycogen phosphorylase of liver and muscle are isozymes with different regulatory mechanisms

• One molecule of epinephrine/glucagon release ~10,000 molecules of glucose

• Differences in last step occurs because glucose-6-phosphatase is only expressed in hepatocytes

29


phosphorylase b kinase, which in turn converts phos-phorylase b to its active a form, initiating the release ofglucose into the blood. When blood glucose levels returnto normal, glucose enters hepatocytes and binds to aninhibitory allosteric site on phosphorylase a. This bind-ing also produces a conformational change that exposes

the phosphorylated Ser residues to PP1, which cat-alyzes their dephosphorylation and inactivates the phos-phorylase (Fig. 15–36). The allosteric site for glucoseallows liver glycogen phosphorylase to act as its ownglucose sensor and to respond appropriately to changesin blood glucose.

Inactiveglycogen

phosphorylase b

Inactivephosphorylase b

kinase

Activephosphorylase b

kinase

Inactive PKA Active PKA

Epinephrine

Gs

ATP

HepatocyteGlucagon

Cyclic AMP20x molecules

x molecules

10x molecules

100x molecules

1,000x molecules

10,000x molecules

10,000x molecules

Activeglycogen

phosphorylase a

Glucose 1-phosphate

[Ca2+]

adenylylcyclase

[AMP]

Glycogen

Glycolysis

Muscle contraction

Glucose

Blood glucose

Myocyte

!

FIGURE 15–35 Cascade mechanism of epinephrine andglucagon action. By binding to specific surface receptors,either epinephrine acting on a myocyte (left) or glucagonacting on a hepatocyte (right) activates a GTP-binding pro-tein Gs! (see Fig. 12–4). Active Gs! triggers a rise in [cAMP],activating PKA. This sets off a cascade of phosphorylations;PKA activates phosphorylase b kinase, which then activatesglycogen phosphorylase. Such cascades effect a large am-plification of the initial signal; the figures in pink boxes areprobably low estimates of the actual increase in number ofmolecules at each stage of the cascade. The resulting break-down of glycogen provides glucose, which in the myocytecan supply ATP (via glycolysis) for muscle contraction andin the hepatocyte is released into the blood to counter thelow blood glucose.

(active)

CH2 O P

Allostericsites empty

2 GlucoseCH2

O

CH2

O

P

phosphorylase aphosphatase

(PP1)

2Pi

Glc

CH2CH2

OH OH

(less active)

Glc Glc Glc

CH2OP

P

Insulin

Phosphorylase a Phosphorylase a Phosphorylase b

FIGURE 15–36 Glycogen phosphorylase of liver as a glucose sensor.Glucose binding to an allosteric site of the phosphorylase a isozyme ofliver induces a conformational change that exposes its phosphorylatedSer residues to the action of phosphorylase a phosphatase (PP1). This

phosphatase converts phosphorylase a to phosphorylase b, sharply re-ducing the activity of phosphorylase and slowing glycogen breakdownin response to high blood glucose. Insulin also acts indirectly to stim-ulate PP1 and slow glycogen breakdown.


Reciprocal Hormonal Regulation of Glycogen Catabolism

Duringrest

Duringexercise

30

• Glycogen phosphorylase b (inactive state) is predominant form in muscle

• Glucagon and epinephrin stimulate activity of phosphorylase kinase A (PKA)

• PKA phosphorylates and activates phosphorylase b kinase

• Phosphorylase b kinase converts phosphorylase b to a

• Phosphorylase a begins digesting glycogen and releases glucose 1-phosphate

• Insulin stimulates PP1. PP1 dephosphorylates phosphorylase a to make phosphorylase b (inactive)


Allosteric Regulation of Glycogen Catabolism

31

• At low [glucose]: phosphorylase a is present (glycogen catabolism)

• At high [glucose]: glucose is greater than Kd of Phosphorylase a for glucose; thus, glucose binds to the allosteric site of Phosphorylase a, induces conformational change to expose the Ser-P residue

• At high [glucose]: insulin is released. Insulin promotes PP1 to dephosphorylate phosphorylase a. Phosphorylase b does not digest glycogen.



Topics


Regulation of Glycogen Synthase

33

• Glycogen synthase a (the default state) makes glycogen

• Glycogen synthase a is inactivated by phosphorylation of hydroxyl side chains by CKII then GSK3

• Insulin inhibits GSK3 and allows glycogen synthesis to continue

• In liver, phosphoprotein phosphatase (PP1) is bound to the glycogen particle and promotes conversion of glycogen synthase b to a

• Glucose 6-P binds to an allosteric site on glycogen synthase b making the enzyme a better substrate for dephosphorylation by PP1 and conversion to glycogen synthase a

glycogen phosphorylase a


Activation of Glycogen Synthesis by Insulin

34


from the three Ser residues phosphorylated by GSK3.Glucose 6-phosphate binds to an allosteric site on glyco-gen synthase b, making the enzyme a better substratefor dephosphorylation by PP1 and causing its activation.By analogy with glycogen phosphorylase, which acts asa glucose sensor, glycogen synthase can be regarded asa glucose 6-phosphate sensor. In muscle, a differentphosphatase may have the role played by PP1 in liver,activating glycogen synthase by dephosphorylating it.

Glycogen Synthase Kinase 3 Mediates Some of the Actions of Insulin

As we saw in Chapter 12, one way in which insulin trig-gers intracellular changes is by activating a protein ki-nase (PKB) that in turn phosphorylates and inactivatesGSK3 (Fig. 15–39; see also Fig. 12–16). Phosphoryla-tion of a Ser residue near the amino terminus of GSK3converts that region of the protein to a pseudosubstrate,which folds into the site at which the priming phospho-rylated Ser residue normally binds (Fig. 15–38b). Thisprevents GSK3 from binding the priming site of a realsubstrate, thereby inactivating the enzyme and tippingthe balance in favor of dephosphorylation of glycogensynthase by PP1. Glycogen phosphorylase can also af-fect the phosphorylation of glycogen synthase: activeglycogen phosphorylase directly inhibits PP1, prevent-ing it from activating glycogen synthase (Fig. 15–37).

Although first discovered in its role in glycogen me-tabolism (hence the name glycogen synthase kinase),GSK3 clearly has a much broader role than the regula-tion of glycogen synthase. It mediates signaling by in-sulin and other growth factors and nutrients, and it actsin the specification of cell fates during embryonic devel-opment. Among its targets are cytoskeletal proteins andproteins essential for mRNA and protein synthesis.These targets, like glycogen synthase, must first un-

dergo a priming phosphorylation by another protein ki-nase before they can be phosphorylated by GSK3.

Phosphoprotein Phosphatase 1 Is Central to Glycogen Metabolism

A single enzyme, PP1, can remove phosphoryl groupsfrom all three of the enzymes phosphorylated in re-sponse to glucagon (liver) and epinephrine (liver andmuscle): phosphorylase kinase, glycogen phosphory-lase, and glycogen synthase. Insulin stimulates glycogensynthesis by activating PP1 and by inactivating GSK3.

Phosphoprotein phosphatase 1 does not exist free inthe cytosol, but is tightly bound to its target proteins by oneof a family of glycogen-targeting proteins that bindglycogen and each of the three enzymes, glycogen phos-phorylase, phosphorylase kinase, and glycogen synthase(Fig. 15–40). PP1 is itself subject to covalent and al-losteric regulation: it is inactivated when phosphorylated byPKA and is allosterically activated by glucose 6-phosphate.

Allosteric and Hormonal Signals Coordinate CarbohydrateMetabolism Globally

Having looked at the mechanisms that regulate individ-ual enzymes, we can now consider the overall shifts incarbohydrate metabolism that occur in the well-fedstate, during fasting, and in the fight-or-flight response—signaled by insulin, glucagon, and epinephrine, respec-tively. We need to contrast two cases in which regulationserves different ends: (1) the role of hepatocytes in sup-plying glucose to the blood, and (2) the selfish use of car-bohydrate fuels by nonhepatic tissues, typified by skeletalmuscle (myocytes), to support their own activities.

After ingestion of a carbohydrate-rich meal, the ele-vation of blood glucose triggers insulin release(Fig. 15–41, top). In a hepatocyte, insulin has two imme-

ActiveInactive

3PiPP1

Cytosol

OHOH

OH

PKB

P

GSK3GSK3

P

P

P

PIP3 PIP2

PDK-1

Insulin Insulinreceptor

OHIRS-1 IRS-1

P

PI-3K

Plasmamembrane

Glycogensynthase

b

Glycogensynthase

a

Inactive

Active

FIGURE 15–39 The path from insulin to GSK3 andglycogen synthase. Insulin binding to its receptoractivates a tyrosine protein kinase in the receptor,which phosphorylates insulin receptor substrate-1(IRS-1). The phosphotyrosine in this protein is thenbound by phosphatidylinositol 3-kinase (PI-3K),which converts phosphatidylinositol 4,5-bisphos-phate (PIP2) in the membrane to phosphatidylinosi-tol 3,4,5-trisphosphate (PIP3). A protein kinase(PDK-1) that is activated when bound to PIP3 acti-vates a second protein kinase (PKB), which phos-phorylates glycogen synthase kinase 3 (GSK3) in itspseudosubstrate region, inactivating it by the mech-anisms shown in Figure 15–38b. The inactivation ofGSK3 allows phosphoprotein phosphatase 1 (PP1) todephosphorylate and thus activate glycogen syn-thase. In this way, insulin stimulates glycogen synthe-sis. (See Fig. 12–16 for more details on insulin action.)

• Insulin binding to receptor activates receptor bound protein Tyr kinase (not shown)

• Kinase phosphorylates IRS-1 (insulin receptor substrate)

• IRS-1-P binds phosphatidylinositol 3-kinase (PI-3K) which converts PIP2 to PIP3

• Membrane embedded PIP3 is a ligand for and activates protein kinase PDK-1

• PDK1 activates protein kinase B (PKB) • PKB phosphorylates glycogen synthase

kinase 3 (GSK3), which is normally ON and inhibiting glycogen synthesis

• GSK3 is now inactive and unable to inactivate glycogen synthesis

• PP1 can now dephosphorylate glycogen synthase b to form glycogen synthase a

• glycogen synthase a can make glycogen

PIP2 PIP3

glycogen catabolism no glycogen catabolism



Topics


Regulation of Carbohydrate Metabolism15.5 Coordinated Regulation of Glycogen Synthesis and Breakdown 607

Phosphorylase kinase

GM

Glycogenphosphorylase Glycogen

synthase

PKA

insulin-sensitivekinase

Inhibitor 1

epinephrine

Glycogengranule

Phosphorylatedinhibitor 1binds andinactivates PP1

insulin

12GM

PP1

PP1

P

GM

P

P

P

P

FIGURE 15–40 Glycogen-targeting protein GM. Theglycogen-targeting protein GM is one of a family of proteinsthat bind other proteins (including PP1) to glycogen parti-cles. GM can be phosphorylated at two different sites in re-sponse to insulin or epinephrine. 1 Insulin-stimulatedphosphorylation of GM site 1 activates PP1, which dephos-phorylates phosphorylase kinase, glycogen phosphorylase,and glycogen synthase (not shown). 2 Epinephrine-stimulated phosphorylation of GM site 2 causes dissoci-ation of PP1 from the glycogen particle, preventing itsaccess to glycogen phosphorylase and glycogen syn-thase. PKA also phosphorylates a protein (inhibitor 1)that, when phosphorylated, inhibits PP1. By these means,insulin inhibits glycogen breakdown and stimulatesglycogen synthesis, and epinephrine (or glucagon in theliver) has the opposite effects.

High bloodglucose

Insulin

Insulin-sensitiveprotein kinase

Phosphorylasekinase

PKB

GSK-3PP1

Glycogenphosphorylase

Glycogenbreakdown

Glycogensynthesis Glycolysis

Glycogenbreakdown

Glycogensynthesis


Phosphorylasekinase

FBPase-2PFK-2

PKA

[cAMP]

Glucagon

Low blood glucose

Pyruvatekinase L

Glycogensynthase

PFK-1

[F26BP]

Glycogensynthase

Synthesis ofhexokinase II,

PFK-1, pyruvatekinase

GLUT2

[Glucose]inside

Glycolysis

FIGURE 15–41 Regulation of carbohydrate metabolismin the liver. Arrows indicate causal relationships betweenthe changes they connect. For example, an arrow from gAto hB means that a decrease in A causes an increase in B.Pink arrows connect events that result from high bloodglucose; blue arrows connect events that result from lowblood glucose.

• Insulin (turns OFF GSK3) • GM serves to anchor proteins to glycogen

particles • Insulin stimulation phosphorylates GM at site-1

to activate it and recruit PP1-complex • PP1 (the reciprocal regulator) is tightly bound

to its target proteins and removes phosphates to promote glycogen synthesis

• Glycogen phosphorylase a to inactivate it and demote glycogen catabolism

• Phosphorylase kinase to inactivate it (phosphorylase kinase activates glycogen phosphorylase a)

• Glycogen synthase b to activate synthesis • Epinephrin stimulates PKA!

• PKA phosphorylate GM site-2 to disassemble PP1-complex from granule

• PKA phosphorylates phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase for glucose 1-P release


Regulation of carbohydrate metabolism

After a meal, [glucose] increases • Glucose is entering hepatocytes via facilitated diffusion (GLUT) • Pancreas releases insulin • Insulin binds receptor of hepatocyte cells • Insulin turns OFF GSK3 • The off state of GSK3 can not inhibit glycogen synthesis • High [glucose] promotes release of hexokinase IV from nucleus and

conversion of glucose to glucose-6-phosphate • Glucose-6-phosphate can enter glycolysis or be converted to glucose-1-

phosphate for glycogen synthesis • Glycogen is ~10% of liver’s weight • Similar things happen in myocytes


Regulation of carbohydrate metabolismBetween meals [glucose] decreases • Pancreas releases glucagon • Glucagon activates PKA -leads to production of glucose 6-phosphate by

glycogen breakdown and gluconeogenesis • PKA

• Promotes glycogen catabolism by phosphorylating and activating phosphorylase kinase which activates glycogen phosphorylase (initiates glycogen catabolism)

• Inhibits glycogen synthesis by phosphorylating and inactivating glycogen synthase

• Phosphorylates PFK-2/FBPase-2 leading to a drop in [F-2,6BP]. F2,6BP is an allosteric activator of PFK-1 (glycolysis) and inactivator of FPBase-1 (gluconeogenesis)

• Phosphorylates and inactivates pyruvate kinase (step 10 of glycolysis) • Glucose 6-phosphate is transported into ER of hepatocytes, dephosphorylated,

transported out of ER and into cytoplasm, and finally transported into blood


During exercise or for flight • Skeletal muscle contains its own glycogen supply and cannot preform

gluconeogenesis • Muscle has no receptor for glucagon • Muscle pyruvate kinase not regulated by PKA, thus always running • Pancreas releases epinephrine stimulates PKA • PKA

• Phosphorylates and activates glycogen phosphorylase kinase, faster glycogen breakdown

• PKA phosphorylate GM site-2 to inactivate, and disassemble PP1-complex from glycogen granule


Regulation of carbohydrate metabolism

Biosignaling424

receptors mediate changes in fuel metabolism, as de-scribed in Chapter 23, including the increased break-down of glycogen and fat. Adrenergic receptors of the!1 and !2 subtypes act through the same mechanism, soin our discussion, “!-adrenergic” applies to both types.

The !-adrenergic receptor is an integral proteinwith seven hydrophobic regions of 20 to 28 amino acidresidues that “snake” back and forth across the plasmamembrane seven times (thus the alternative names forGPCRs: serpentine receptors or heptahelical re-ceptors). The binding of epinephrine to a site on the re-ceptor deep within the plasma membrane (Fig. 12–4a,step 1 ) promotes a conformational change in the re-ceptor’s intracellular domain that affects its interactionwith the second protein in the signal-transduction path-way, stimulatory G protein, or GS, on the cytoplasmicside of the membrane. Alfred G. Gilman and Martin Rod-

bell discovered that active Gs stimulates the productionof cAMP (cyclic AMP) by adenylyl cyclase (see below)in the plasma membrane. Gs, the prototype for a familyof G proteins that act in biosignaling (see Box 12–2), isheterotrimeric, with subunit structure "!#. When thenucleotide-binding site of Gs (on the " subunit) is occu-pied by GTP, Gs is turned on and can activate adenylylcyclase (AC in Fig. 12–4a); with GDP bound to the site,Gs is switched off. The activated !-adrenergic receptorinteracts with Gs, catalyzing replacement of bound GDPwith GTP and converting Gs to its active form (step 2 ).As this occurs, the ! and # subunits of Gs dissociatefrom the " subunit as a !# dimer, and Gs", with itsbound GTP, moves in the plane of the membrane fromthe receptor to a nearby molecule of adenylyl cyclase(step 3 ). Gs" is held to the membrane by a covalentlyattached palmitoyl group (see Fig. 11–14).

FIGURE 12–4 Transduction of the epinephrine signal: the !-adrenergicpathway. (a)The mechanism that couples binding of epinephrine (E) to itsreceptor (Rec) with activation of adenylyl cyclase (AC); the seven stepsare discussed further in the text. The same adenylyl cyclase molecule inthe plasma membrane may be regulated by a stimulatory G protein (Gs),

as shown, or an inhibitory G protein (Gi, not shown). Gs and Gi are un-der the influence of different hormones. Hormones that induce GTPbinding to Gi cause inhibition of adenylyl cyclase, resulting in lower cel-lular [cAMP]. (b) The combined action of the enzymes that catalyzesteps 4 and 7 , forming then inactivating the second messenger, cAMP.

2The occupied receptorcauses replacement ofthe GDP bound to Gsby GTP, activating Gs.

1Epinephrine binds toits specific receptor.

ATP

cAMP

5!-AMP

cyclic nucleotidephosphodiesterase

GTP GDP

5cAMPactivatesPKA.

6Phosphorylation ofcellular proteins byPKA causes thecellular response toepinephrine.

Outside

Inside

7cAMP is degraded,reversing theactivation of PKA.

E

NH3

"OOC

#

Rec$

%&

Gs

Gs

GDP

GTP

AC

%

4Adenylyl cyclasecatalyzes theformation of cAMP.

3Gs ( subunit) movesto adenylyl cyclaseand activates it.

%

(a)

"O P PO

O" O"

O O

H

P

O"

O

OO

HH

H

N

O

OH

O

H

NN

N

CH

NH2

2

ATP Adenosine 5! -monophosphate (AMP)

H

P

O"

OO

HH

H

N

O

O

O

H

NN

N

CH2

5!"

H

P

O"

O

OO

HH

H

N

O

OH

O

H

NN

N

CH

NH2

2

5!

Adenosine 3!,5!-cyclicmonophosphate

(cAMP)

NH2

3! 3!

PPi

adenylylcyclase

H2O

cyclicnucleotide

phosphodiesterase

(b)

Regulation of Carbohydrate Metabolism in Liver


from the three Ser residues phosphorylated by GSK3.Glucose 6-phosphate binds to an allosteric site on glyco-gen synthase b, making the enzyme a better substratefor dephosphorylation by PP1 and causing its activation.By analogy with glycogen phosphorylase, which acts asa glucose sensor, glycogen synthase can be regarded asa glucose 6-phosphate sensor. In muscle, a differentphosphatase may have the role played by PP1 in liver,activating glycogen synthase by dephosphorylating it.

Glycogen Synthase Kinase 3 Mediates Some of the Actions of Insulin

As we saw in Chapter 12, one way in which insulin trig-gers intracellular changes is by activating a protein ki-nase (PKB) that in turn phosphorylates and inactivatesGSK3 (Fig. 15–39; see also Fig. 12–16). Phosphoryla-tion of a Ser residue near the amino terminus of GSK3converts that region of the protein to a pseudosubstrate,which folds into the site at which the priming phospho-rylated Ser residue normally binds (Fig. 15–38b). Thisprevents GSK3 from binding the priming site of a realsubstrate, thereby inactivating the enzyme and tippingthe balance in favor of dephosphorylation of glycogensynthase by PP1. Glycogen phosphorylase can also af-fect the phosphorylation of glycogen synthase: activeglycogen phosphorylase directly inhibits PP1, prevent-ing it from activating glycogen synthase (Fig. 15–37).

Although first discovered in its role in glycogen me-tabolism (hence the name glycogen synthase kinase),GSK3 clearly has a much broader role than the regula-tion of glycogen synthase. It mediates signaling by in-sulin and other growth factors and nutrients, and it actsin the specification of cell fates during embryonic devel-opment. Among its targets are cytoskeletal proteins andproteins essential for mRNA and protein synthesis.These targets, like glycogen synthase, must first un-

dergo a priming phosphorylation by another protein ki-nase before they can be phosphorylated by GSK3.

Phosphoprotein Phosphatase 1 Is Central to Glycogen Metabolism

A single enzyme, PP1, can remove phosphoryl groupsfrom all three of the enzymes phosphorylated in re-sponse to glucagon (liver) and epinephrine (liver andmuscle): phosphorylase kinase, glycogen phosphory-lase, and glycogen synthase. Insulin stimulates glycogensynthesis by activating PP1 and by inactivating GSK3.

Phosphoprotein phosphatase 1 does not exist free inthe cytosol, but is tightly bound to its target proteins by oneof a family of glycogen-targeting proteins that bindglycogen and each of the three enzymes, glycogen phos-phorylase, phosphorylase kinase, and glycogen synthase(Fig. 15–40). PP1 is itself subject to covalent and al-losteric regulation: it is inactivated when phosphorylated byPKA and is allosterically activated by glucose 6-phosphate.

Allosteric and Hormonal Signals Coordinate CarbohydrateMetabolism Globally

Having looked at the mechanisms that regulate individ-ual enzymes, we can now consider the overall shifts incarbohydrate metabolism that occur in the well-fedstate, during fasting, and in the fight-or-flight response—signaled by insulin, glucagon, and epinephrine, respec-tively. We need to contrast two cases in which regulationserves different ends: (1) the role of hepatocytes in sup-plying glucose to the blood, and (2) the selfish use of car-bohydrate fuels by nonhepatic tissues, typified by skeletalmuscle (myocytes), to support their own activities.

After ingestion of a carbohydrate-rich meal, the ele-vation of blood glucose triggers insulin release(Fig. 15–41, top). In a hepatocyte, insulin has two imme-

ActiveInactive

3PiPP1

Cytosol

OHOH

OH

PKB

P

GSK3GSK3

P

P

P

PIP3 PIP2

PDK-1

Insulin Insulinreceptor

OHIRS-1 IRS-1

P

PI-3K

Plasmamembrane

Glycogensynthase

b

Glycogensynthase

a

Inactive

Active

FIGURE 15–39 The path from insulin to GSK3 andglycogen synthase. Insulin binding to its receptoractivates a tyrosine protein kinase in the receptor,which phosphorylates insulin receptor substrate-1(IRS-1). The phosphotyrosine in this protein is thenbound by phosphatidylinositol 3-kinase (PI-3K),which converts phosphatidylinositol 4,5-bisphos-phate (PIP2) in the membrane to phosphatidylinosi-tol 3,4,5-trisphosphate (PIP3). A protein kinase(PDK-1) that is activated when bound to PIP3 acti-vates a second protein kinase (PKB), which phos-phorylates glycogen synthase kinase 3 (GSK3) in itspseudosubstrate region, inactivating it by the mech-anisms shown in Figure 15–38b. The inactivation ofGSK3 allows phosphoprotein phosphatase 1 (PP1) todephosphorylate and thus activate glycogen syn-thase. In this way, insulin stimulates glycogen synthe-sis. (See Fig. 12–16 for more details on insulin action.)

15.5 Coordinated Regulation of Glycogen Synthesis and Breakdown 607

Phosphorylase kinase

GM

Glycogenphosphorylase Glycogen

synthase

PKA

insulin-sensitivekinase

Inhibitor 1

epinephrine

Glycogengranule

Phosphorylatedinhibitor 1binds andinactivates PP1

insulin

12GM

PP1

PP1

P

GM

P

P

P

P

FIGURE 15–40 Glycogen-targeting protein GM. Theglycogen-targeting protein GM is one of a family of proteinsthat bind other proteins (including PP1) to glycogen parti-cles. GM can be phosphorylated at two different sites in re-sponse to insulin or epinephrine. 1 Insulin-stimulatedphosphorylation of GM site 1 activates PP1, which dephos-phorylates phosphorylase kinase, glycogen phosphorylase,and glycogen synthase (not shown). 2 Epinephrine-stimulated phosphorylation of GM site 2 causes dissoci-ation of PP1 from the glycogen particle, preventing itsaccess to glycogen phosphorylase and glycogen syn-thase. PKA also phosphorylates a protein (inhibitor 1)that, when phosphorylated, inhibits PP1. By these means,insulin inhibits glycogen breakdown and stimulatesglycogen synthesis, and epinephrine (or glucagon in theliver) has the opposite effects.

High bloodglucose

Insulin

Insulin-sensitiveprotein kinase

Phosphorylasekinase

PKB

GSK-3PP1


Glycogenbreakdown

Glycogensynthesis Glycolysis

Glycogenbreakdown

Glycogensynthesis


Phosphorylasekinase

FBPase-2PFK-2

PKA

[cAMP]

Glucagon

Low blood glucose

Pyruvatekinase L

Glycogensynthase

PFK-1

[F26BP]

Glycogensynthase

Synthesis ofhexokinase II,

PFK-1, pyruvatekinase

GLUT2

[Glucose]inside

Glycolysis

FIGURE 15–41 Regulation of carbohydrate metabolismin the liver. Arrows indicate causal relationships betweenthe changes they connect. For example, an arrow from gAto hB means that a decrease in A causes an increase in B.Pink arrows connect events that result from high bloodglucose; blue arrows connect events that result from lowblood glucose.


Carbohydrate Metabolism in Liver and Muscle

• The liver reacts to both glucagon and epinephrine • Glycogen synthesis • Glycogen catabolism for increased blood [glucose] • Gluconeogenesis for increased blood [glucose]

• Muscle reacts to epinephrine (no glucagon receptor) • Glycolysis for the ATP it needs for muscle contraction


biochemistry - i · biochemistry - i mondays and ... • ∆g’o, biochemical standard free...

Documents