Biochemistry, 4th Edition P71 pot

The final step to produce glucose, hydrolysis of glucose-6-phosphate, is mediated ATP ATP Glucose + Glucose-6-P Fructose-6-P H2O H2O Fructose-1,6-bisP Glyceraldehyde-3-P Dihydroxyacetone

esis are the liver and kidneys, which account for about 90% and 10% of the body’s

gluconeogenic activity, respectively Glucose produced by gluconeogenesis in the

liver and kidneys is released into the blood and is subsequently absorbed by brain,

heart, muscle, and red blood cells to meet their metabolic needs In turn, pyruvate

and lactate produced in these tissues are returned to the liver and kidneys to be

used as gluconeogenic substrates

Gluconeogenesis Is Not Merely the Reverse of Glycolysis

In some ways, gluconeogenesis is the reverse, or antithesis, of glycolysis Glucose

is synthesized, not catabolized; ATP is consumed, not produced; and NADH is

ox-idized to NAD, rather than the other way around However, gluconeogenesis

cannot be merely the reversal of glycolysis, for two reasons First, glycolysis is

exer-gonic, with a G° of approximately 74 kJ/mol If gluconeogenesis were merely

the reverse, it would be a strongly endergonic process and could not occur

spon-taneously Somehow the energetics of the process must be augmented so that

glu-coneogenesis can proceed spontaneously Second, the processes of glycolysis and

gluconeogenesis must be regulated in a reciprocal fashion so that when glycolysis

is active, gluconeogenesis is inhibited, and when gluconeogenesis is proceeding,

glycolysis is turned off Both of these limitations are overcome by having unique

reactions within the routes of glycolysis and gluconeogenesis, rather than a

com-pletely shared pathway

Gluconeogenesis—Something Borrowed, Something New

The complete route of gluconeogenesis is shown in Figure 22.1, side by side with the

glycolytic pathway Gluconeogenesis employs four different reactions, catalyzed by

four different enzymes, for the three steps of glycolysis that are highly exergonic

HUMAN BIOCHEMISTRY

The Chemistry of Glucose Monitoring Devices

Individuals with diabetes must measure their serum glucose

con-centration frequently, often several times a day The advent of

com-puterized, automated devices for glucose monitoring has made this

necessary chore easier, far more accurate, and more convenient

than it once was These devices all use a simple chemical scheme for

glucose measurement that involves oxidation of glucose to gluconic

acid by glucose oxidase This reaction produces two molecules of

hydrogen peroxide per molecule of glucose oxidized The H2O2is

then used to oxidize a dye, such as o -dianisidine, to a colored

prod-uct that can be measured:

Glucose 2 H2O O2⎯⎯→ gluconic acid  2 H2O2

o -dianisidine (colorless)  H2O2⎯⎯→ oxidized o-anisidine

(colored) H2O The amount of colored dye produced is directly proportional to

the amount of glucose in the sample

The patient typically applies a drop of blood (from a

finger-prick*) to a plastic test strip that is then inserted into the glucose

monitor Within half a minute, a digital readout indicates the

blood glucose value Modern glucose monitors store several days

of glucose measurements, and the data can be easily transferred to

a computer for analysis and graphing

*How does the monitor deal with getting just the right amount of blood?

The blood flows up an absorbent “wick” by capillary action It is impossible

to overfill this device, but the monitor will give an error signal if not

enough blood flows up the strip.

(and highly regulated) In essence, seven of the ten steps of glycolysis are merely re-versed in gluconeogenesis The six reactions between fructose-1,6-bisphosphate and PEP are shared by the two pathways, as is the isomerization of glucose-6-P to fructose-6-P The three exergonic, regulated reactions—the hexokinase (glucokinase), phos-phofructokinase, and pyruvate kinase reactions—are replaced by alternative reac-tions in the gluconeogenic pathway

The conversion of pyruvate to PEP that initiates gluconeogenesis is

accom-plished by two unique reactions Pyruvate carboxylase catalyzes the first, convert-ing pyruvate to oxaloacetate Then, PEP carboxykinase catalyzes the conversion

of oxaloacetate to PEP Conversion of fructose-1,6-bisphosphate to

fructose-6-phosphate is catalyzed by a specific phosphatase, fructose-1,6-bisphosphatase.

The final step to produce glucose, hydrolysis of glucose-6-phosphate, is mediated

ATP

ATP

Glucose

+

Glucose-6-P

Fructose-6-P

H2O

H2O

Fructose-1,6-bisP

Glyceraldehyde-3-P Dihydroxyacetone-P

Glycerol 1,3-Bisphosphoglycerate

3-Phosphoglycerate

2-Phosphoglycerate

PEP

Pyruvate

Lactate

Oxaloacetate

Amino acids

This reaction occurs in the ER

Mitochondrial matrix

ATP ATP

ATP

ATP

ADP Pi

Pi

Pi

ADP ADP

ADP ADP

GDP

CO2

NAD +

NADH

NAD +

NADH

Glucose-6-phosphatase

Fructose-1,6-bisphosphatase

Pyruvate carboxylase PEP carboxykinase

FIGURE 22.1 The pathways of gluconeogenesis and

glycolysis Species in blue, green, and peach-colored

shaded boxes indicate other entry points for

gluconeo-genesis (in addition to pyruvate).

by glucose-6-phosphatase Each of these steps is considered in detail in the

fol-lowing paragraphs The overall conversion of pyruvate to PEP by pyruvate

car-boxylase and PEP carboxykinase has a G° close to zero but is pulled along by

sub-sequent reactions The conversion of fructose-1,6-bisphosphate to glucose in the

last three steps of gluconeogenesis is strongly exergonic, with a G° of about

30.5 kJ/mol This sequence of two phosphatase reactions separated by an

iso-merization accounts for most of the free energy release that makes the

gluconeo-genesis pathway spontaneous

Four Reactions Are Unique to Gluconeogenesis

1 Pyruvate Carboxylase—A Biotin-Dependent Enzyme Initiation of

gluconeo-genesis occurs in the pyruvate carboxylase reaction—the conversion of pyruvate to

oxaloacetate

The reaction takes place in two discrete steps, involves ATP and bicarbonate as

sub-strates, and utilizes biotin as a coenzyme and acetyl-CoA as an allosteric activator

Pyruvate carboxylase is a tetrameric enzyme (with a molecular mass of about

500 kD) Each monomer possesses a biotin covalently linked to the

of a lysine residue at the active site (Figure 22.2) The first step of the reaction

involves nucleophilic attack of a bicarbonate oxygen at the -P of ATP to form

carbonylphosphate with biotin occurs rapidly to form N-carboxybiotin, liberating

inorganic phosphate The third step involves abstraction of a proton from the C-3

of pyruvate, forming a carbanion that can attack the carbon of N-carboxybiotin to

form oxaloacetate

Pyruvate Carboxylase Is Allosterically Activated by Acetyl-Coenzyme A Two

particu-larly interesting aspects of the pyruvate carboxylase reaction are (1) allosteric

activa-tion of the enzyme by acyl-CoA derivatives and (2) compartmentaactiva-tion of the reacactiva-tion

CH3C

O

+ HCO3– +

CH2C –OOC

O COO–

Oxaloacetate

ADP Pi

CH2CH2CH2CH2 NH

C

CH2

CH2

CH2

CH2

S

H N

N H O

Lysine

Biotin

O

E

FIGURE 22.2 Covalent linkage of biotin to an active-site lysine in pyruvate carboxylase.

In most organisms, pyruvate carboxylase is a homo-tetramer of 130-kD subunits, with each subunit com-posed of three functional domains named biotin car-boxylase, carboxyl transferase, and biotin carboxyl carrier protein Shown here is the biotin carboxylase domain of

pyruvate carboxylase from Bacillus thermodenitrificans.

(pdb id  2DZD).

–O

C

O

HO

P

O

O O–

O P

O

O–

O P

O

O–

O–

C O

P

O

O

O–

O

N NH

S O

HN NH

S

–O –O

–O

C O

–O C O

–CH2 C COO–

O

CH2 C COO–

O

Lys

CH2 C COO–

O

B E Biotin

Adenosine

FIGURE 22.3 A mechanism for the pyruvate carboxylase reaction Bicarbonate must be activated for attack

by the pyruvate carbanion This activation is driven by ATP and involves formation of a carbonylphosphate

intermediate—a mixed anhydride of carbonic and phosphoric acids (Carbonylphosphate and

carboxyphos-phate are synonyms.)

in the mitochondrial matrix The carboxylation of biotin requires the presence (at

an allosteric site) of acetyl-CoA or other acylated CoA derivatives The second half of the carboxylase reaction—the attack by pyruvate to form oxaloacetate—is not af-fected by CoA derivatives

Activation of pyruvate carboxylase by acetyl-CoA provides an important physio-logical regulation Acetyl-CoA is the primary substrate for the TCA cycle, and oxa-loacetate (formed by pyruvate carboxylase) is an important intermediate in both the TCA cycle and the gluconeogenesis pathway If levels of ATP and/or acetyl-CoA (or other acyl-CoAs) are low, pyruvate is directed primarily into the TCA cycle, which eventually promotes the synthesis of ATP If ATP and acetyl-CoA levels are high, pyruvate is converted to oxaloacetate and consumed in gluconeogenesis Clearly, high levels of ATP and CoA derivatives are signs that energy is abundant and that metabolites will be converted to glucose (and perhaps even glycogen) If the energy status of the cell is low (in terms of ATP and CoA derivatives), pyruvate

is consumed in the TCA cycle Also, as noted in Chapter 19, pyruvate carboxylase is

an important anaplerotic enzyme Its activation by acetyl-CoA leads to oxaloacetate formation, replenishing the level of TCA cycle intermediates

Compartmentalized Pyruvate Carboxylase Depends on Metabolite Conversion and Transport The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria By contrast, the next enzyme in the gluco-neogenic pathway, PEP carboxykinase, may be localized in the cytosol, in the mito-chondria, or both For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria Pyruvate is trans-ported into the mitochondrial matrix (Figure 22.4), where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis; see Figure 24.1) Alternatively, it may be converted directly to OAA by pyruvate carboxy-lase and used in gluconeogenesis In tissues where PEP carboxykinase is found only

in the mitochondria, oxaloacetate is converted to PEP, which is then transported to the cytosol for gluconeogenesis However, in tissues that must convert some oxa-loacetate to PEP in the cytosol, a problem arises Oxaoxa-loacetate cannot be transported directly across the mitochondrial membrane Instead, it must first be transformed into malate or aspartate for transport across the mitochondrial inner membrane (Figure 22.4) Cytosolic malate and aspartate must be reconverted to oxaloacetate before continuing along the gluconeogenic route

2 PEP Carboxykinase The second reaction in the gluconeogenic pyruvate–PEP bypass is the conversion of oxaloacetate to PEP

Production of a high-energy metabolite such as PEP requires energy The energetic requirements are handled in two ways here First, the CO2added to pyruvate in the pyruvate carboxylase step is removed in the PEP carboxykinase reaction Decarboxy-lation is a favorable process and helps drive the formation of the very high-energy enol phosphate in PEP This decarboxylation drives a reaction that would otherwise

be highly endergonic Note the inherent metabolic logic in this pair of reactions: Pyruvate carboxylase consumed an ATP to drive a carboxylation so that the PEP carboxykinase could use the decarboxylation to facilitate formation of PEP Second, another high-energy phosphate is consumed by the carboxykinase Mammals and several other species use GTP in this reaction, rather than ATP The use of GTP here

is equivalent to the consumption of an ATP, due to the activity of the nucleoside

is crucial to the synthesis of PEP in this step The overall G for the pyruvate

car-boxylase and PEP carboxykinase reactions under physiological conditions in the liver

C

CH2

C

H2C

COO–

Oxaloacetate

+

PEP carboxykinase

PEP

NAD +

NADH

Pyruvate

Malate

Pyruvate

Oxaloacetate

Malate

Oxaloacetate

Gluconeogenesis

NADH

NAD+

FIGURE 22.4 Pyruvate carboxylase is a

compartmental-ized reaction Pyruvate is converted to oxaloacetate in

the mitochondria Because oxaloacetate cannot be

trans-ported across the mitochondrial membrane, it must be

reduced to malate, transported to the cytosol, and then

oxidized back to oxaloacetate before gluconeogenesis

can continue.

PEP carboxykinase from Escherichia coli with ADP (blue),

pyruvate (purple), and Mg2(pdb id  1OS1).

Go to CengageNOW and click

CengageInteractive to learn more about the pyruvate

carboxylase reaction.

is22.6 kJ/mol Once PEP is formed in this way, the phosphoglycerate mutase,

phoglycerate kinase, glyceraldehyde-3-P dehydrogenase, aldolase, and triose

phos-phate isomerase reactions act to eventually form fructose-1,6-bisphosphos-phate, as shown

in Figure 22.1

3 Fructose-1,6-Bisphosphatase The hydrolysis of fructose-1,6-bisphosphate to

fructose-6-phosphate,

like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic)

reaction under standard-state conditions (G°  16.7 kJ/mol) Under

physio-logical conditions in the liver, the reaction is also exergonic (G 8.6 kJ/mol).

stimu-lates bisphosphatase activity, but fructose-2,6-bisphosphate is a potent allosteric

in-hibitor AMP also inhibits the bisphosphatase; the inhibition by AMP is enhanced

by fructose-2,6-bisphosphate

4 Glucose-6-Phosphatase The final step in the gluconeogenesis pathway is

the conversion of phosphate to glucose by the action of

endo-plasmic reticulum of liver and kidney cells but is absent in muscle and brain For

this reason, gluconeogenesis is not carried out in muscle and brain The

glucose-6-phosphatase system includes the phosphatase itself and three transport proteins,

T1, T2, and T3 The glucose-6-phosphate transporter (T1) takes

glucose-6-phos-phate into the endoplasmic reticulum, where it is hydrolyzed to glucose and Pi

The T2 and T3 transporters export glucose and Pi, respectively, to the cytosol, and

glucose is then exported (to the circulation) by the GLUT2 transporter The

glucose-6-phosphatase reaction involves a phosphorylated enzyme intermediate,

phosphohistidine (Figure 22.6) The G for the glucose-6-phosphatase reaction in

liver is 5.1 kJ/mol

O

H HO

HO H

O3POH2C CH2OPO3

+

O

H HO

HO H

O3POH2C CH2OH

+

P

H2O

Fructose-6-phosphate Fructose-1,6-bisphosphate ΔG⬚ = –16.7 kJ/mol

Fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase from pig with fructose-6-phosphate (orange), AMP (blue), and Mg2(dark blue) (pdb id  1FBP).

Glucose-6-P

Glucose+ Pi

Pi transporter

GLUT2 transporter

Glucose transporter

Glucose-6-phosphatase

Glucose-6-phosphate G-6-P

transporter

Cytosol

ER membrane

ER lumen

T1

T2 T3

Plasma membrane

FIGURE 22.5 Glucose-6-phosphatase is localized in the endoplasmic reticulum Conversion of glucose-6-phosphate to glucose occurs following transport into the endoplasmic reticulum Glucose-6-phosphatase and the three transporters, T1, T2, and T3, are known collec-tively as the glucose-6-phosphatase system.

Coupling with Hydrolysis of ATP and GTP Drives Gluconeogenesis The net reaction for the conversion of pyruvate to glucose in gluconeogenesis is

2 Pyruvate  4 ATP  2 GTP  2 NADH  2 H 6 H2O⎯⎯→

glucose 4 ADP  2 GDP  6 Pi 2 NAD The net free energy change, G°, for this conversion is 37.7 kJ/mol The

con-sumption of a total of six nucleoside triphosphates drives this process forward If glycolysis were merely reversed to achieve the net synthesis of glucose from pyru-vate, the net reaction would be

2 Pyruvate  2 ATP  2 NADH  2 H 2 H2O⎯⎯→

glucose 2 ADP  2 Pi 2 NAD

E

O P O–

O–

O

O OH

OH HO

H2C

CHOH

O–

O

O OH

OH HO

CH2OH

CHOH

N NH +

E

N NH HOPO3– +

H B H

O

FIGURE 22.6 The glucose-6-phosphatase reaction

in-volves formation of a phosphohistidine intermediate.

HUMAN BIOCHEMISTRY

Gluconeogenesis Inhibitors and Other Diabetes Therapy Strategies

Diabetes, the inability to assimilate and metabolize blood glucose,

afflicts millions of people People with type 1 diabetes are unable

to synthesize and secrete insulin On the other hand, people with

type 2 diabetes make sufficient insulin, but the molecular

path-ways that respond to insulin are defective Many type 2 diabetic

people exhibit a condition termed insulin resistance even before

the onset of diabetes Metformin (see accompanying figure) is a

drug that improves sensitivity to insulin, primarily by stimulating

glucose uptake by glucose transporters in peripheral tissues It also

increases binding of insulin to insulin receptors, stimulates

tyro-sine kinase activity (see Chapter 32) of the insulin receptor, and

inhibits gluconeogenesis in the liver

Gluconeogenesis inhibitors may be the next wave in diabetes therapy Drugs that block gluconeogenesis without affecting glycol-ysis would need to target one of the enzymes unique to gluconeo-genesis 3-Mercaptopicolinate and hydrazine inhibit PEP

carboxyk-inase, and chlorogenic acid, a natural product found in the skin of

peaches, inhibits the transport activity of the glucose-6-phosphatase system (but not the glucose-6-phosphatase enzyme activity) The drug S-3483, a derivative of chlorogenic acid, also inhibits the glu-cose-6-phosphatase transport activity and binds a thousand times more tightly to the transporter than chlorogenic acid Drugs of this type may be useful in the treatment of type 2 diabetes

CH3

H3C

H

H2N NH2

NH2

NH NH

HO

HO

OH

O

O

O

O C

OH

Cl

HS N COO –

HO

HO

HO

OH

O

O

O C

OH OH

S-3483 Chlorogenic acid

and the overall G° would be about 74 kJ/mol Such a process would be highly

endergonic and therefore thermodynamically unfeasible Hydrolysis of four

addi-tional high-energy phosphate bonds makes gluconeogenesis thermodynamically

favorable Under physiological conditions, however, gluconeogenesis is somewhat

less favorable than at standard state, with an overall G of 15.6 kJ/mol for the

conversion of pyruvate to glucose

Lactate Formed in Muscles Is Recycled to Glucose in the Liver A final point on the

re-distribution of lactate and glucose in the body serves to emphasize the metabolic

in-teractions between organs Vigorous exercise can lead to oxygen shortage

(anaero-bic conditions), and energy requirements must be met by increased levels of

glycolysis Under such conditions, glycolysis converts NADto NADH, yet O2is

un-available for regeneration of NADvia cellular respiration Instead, large amounts

of NADH are reoxidized by the reduction of pyruvate to lactate The lactate thus

produced can be transported from muscle to the liver, where it is reoxidized by liver

lactate dehydrogenase to yield pyruvate, which is converted eventually to glucose In

this way, the liver shares in the metabolic stress created by vigorous exercise It

ex-ports glucose to muscle, which produces lactate, and lactate from muscle can be

processed by the liver into new glucose This is referred to as the Cori cycle (Figure

22.7) Liver, with a typically high NAD/NADH ratio (about 700), readily produces

more glucose than it can use Muscle that is vigorously exercising will enter

anaer-obiosis and show a decreasing NAD/NADH ratio, which favors reduction of

pyru-vate to lactate

22.2 How Is Gluconeogenesis Regulated?

Nearly all of the reactions of glycolysis and gluconeogenesis take place in the

cyto-sol If metabolic control were not exerted over these reactions, glycolytic

degrada-tion of glucose and gluconeogenic synthesis of glucose could operate

simultane-ously, with no net benefit to the cell and with considerable consumption of ATP

This is prevented by a sophisticated system of reciprocal control, which inhibits

glycolysis when gluconeogenesis is active, and vice versa Reciprocal regulation of

2 NTP Glucose

Pyruvate

LDH Lactate Muscle

Liver

(low [NAD+])

[NADH]

6 NTP

Gluconeogenesis

Glycolysis

Glucose

Pyruvate

LDH Lactate

(high [NAD+])

[NADH]

Blood

NADH

NAD+

NADH

NAD+

FIGURE 22.7 The Cori cycle.

these two pathways depends largely on the energy status of the cell When the en-ergy status of the cell is low, glucose is rapidly degraded to produce needed enen-ergy When the energy status is high, pyruvate and other metabolites are utilized for syn-thesis (and storage) of glucose Moreover, when blood glucose levels are low, gluco-neogenesis is active

In glycolysis, the three regulated enzymes are those catalyzing the strongly exergonic reactions: hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase As noted, the gluconeogenic pathway replaces these three reactions with corresponding reactions that are exergonic in the direction of glucose synthesis: glucose-6-phosphatase, fructose-1,6-bisphosphatase, and the pyruvate carboxylase– PEP carboxykinase pair, respectively These are the three most appropriate sites of regulation in gluconeogenesis

Gluconeogenesis Is Regulated by Allosteric and Substrate-Level Control Mechanisms

The mechanisms of regulation of gluconeogenesis are shown in Figure 22.8 Control

is exerted at all of the predicted sites, but in different ways Glucose-6-phosphatase is

not under allosteric control However, the K mfor the substrate, glucose-6-phosphate,

is considerably higher than the normal range of substrate concentrations As a result, glucose-6-phosphatase displays a near-linear dependence of activity on

sub-strate concentrations and is thus said to be under subsub-strate-level control by

glucose-6-phosphate

Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis It al-losterically inhibits pyruvate kinase (as noted in Chapter 18) and activates pyruvate carboxylase Because it also allosterically inhibits pyruvate dehydrogenase (the en-zymatic link between glycolysis and the TCA cycle), the cellular fate of pyruvate is strongly dependent on acetyl-CoA levels A rise in [acetyl-CoA] indicates that cellu-lar energy levels are high and that carbon metabolites can be directed to glucose synthesis and storage When acetyl-CoA levels drop, the activities of pyruvate kinase and pyruvate dehydrogenase increase and flux through the TCA cycle increases, providing needed energy for the cell

Fructose-1,6-bisphosphatase is another important site of gluconeogenic regula-tion This enzyme is inhibited by AMP and activated by citrate These effects by AMP and citrate are the opposites of those exerted on phosphofructokinase in glycolysis, providing another example of reciprocal regulatory effects When AMP levels in-crease, gluconeogenic activity is diminished and glycolysis is stimulated An increase

in citrate concentration signals that TCA cycle activity can be curtailed and that pyruvate should be directed to sugar synthesis instead

Fructose-2,6-Bisphosphate—Allosteric Regulator of Gluconeogenesis Emile Van Schaftingen and Henri-Géry Hers demonstrated in 1980 that

fructose-2,6-CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

The Pioneering Studies of Carl and Gerty Cori

The Cori cycle is named for Carl and Gerty Cori, who received the

Nobel Prize in Physiology or Medicine in 1947 for their studies of

glycogen metabolism and blood glucose regulation Carl

Ferdi-nand Cori and Gerty Theresa Radnitz were both born in Prague

(then in Austria) They earned medical degrees from the German

University of Prague in 1920 and were married later that year

They joined the faculty of the Washington University School of

Medicine in St Louis in 1931 Their remarkable collaboration

re-sulted in many fundamental advances in carbohydrate and

glyco-gen metabolism They were credited with the discovery of

glucose-1-phosphate, also known at the time as the “Cori ester.” They also

showed that glucose-6-phosphate was produced from glucose-1-P

by the action of phosphoglucomutase They isolated and crystal-lized glycogen phosphorylase and elucidated the pathway of glyco-gen breakdown In 1952, they showed that absence of glucose-6-phosphatase in the liver was the enzymatic defect in von Gierke’s disease, an inherited glycogen-storage disease Six eventual Nobel laureates received training in their laboratory Gerty Cori was the first American woman to receive a Nobel Prize Carl Cori said of their remarkable collaboration: “Our efforts have been largely complementary and one without the other would not have gone

so far…”

CH2

H HO

CH2OH H

O OPO

3

H OH

Fructose-2,6-bisphosphate

bisphosphate is a potent stimulator of phosphofructokinase (see Chapter 18)

Cog-nizant of the reciprocal nature of regulation in glycolysis and gluconeogenesis,

Van Schaftingen and Hers also considered the possibility of an opposite effect—

inhibition—for 1,6-bisphosphatase In 1981, they reported that

fructose-2,6-bisphosphate was indeed a powerful inhibitor of fructose-1,6-bisphosphatase

(Figure 22.9) Inhibition occurs in either the presence or absence of AMP, and the

effects of AMP and fructose-2,6-bisphosphate are synergistic

Cellular levels of fructose-2,6-bisphosphate are controlled by

pathway, and by fructose-2,6-bisphosphatase (F-2,6-BPase) Remarkably, these two

enzymatic activities are both found in the same protein molecule, which is an

exam-ple of a bifunctional, or tandem, enzyme (Figure 22.10) The opposing activities

of this bifunctional enzyme are themselves regulated in two ways First,

fructose-6-phosphate, the substrate of phosphofructokinase and the product of

fructose-1,6-bisphosphatase, allosterically activates PFK-2 and inhibits F-2,6-BPase Second, the

phosphorylation by cAMP-dependent protein kinase of a single Ser (Ser32) residue on

the liver enzyme exerts reciprocal control of the PFK-2 and F-2,6-BPase activities

Phos-phorylation inhibits PFK-2 activity (by increasing the K mfor fructose-6-phosphate) and

stimulates F-2,6-BPase activity

To bloodstream

Glucose

Glucose-6-phosphate

Fructose-6-phosphate

Fructose-1,6-bisphosphate

Phosphoenolpyruvate

Oxaloacetate

Pyruvate

Glucose-6-phosphatase Hexokinase

Fructose-1,6-bisphosphatase Phosphofructokinase

Phosphoenolpyruvate carboxykinase Pyruvate

kinase

Pyruvate carboxylase

Glucose-6-phosphate

Fructose-2,6-bisphosphate

AMP

ATP

Citrate

F-1,6-BP

Acetyl-CoA

ATP

Alanine

cAMP-dependent

phosphorylation

[Glucose-6-phosphate]

(substrate level control)

F-2,6-BP AMP

Acetyl-CoA

Regulation of

glycolysis

Regulation of gluconeogenesis

–

+

+

–

–

+

–

–

–

–

– –

+

FIGURE 22.8 The principal regulatory mechanisms in glycolysis and gluconeogenesis Activators are indicated

by plus signs and inhibitors by minus signs.

Substrate Cycles Provide Metabolic Control Mechanisms

If fructose-1,6-bisphosphatase and phosphofructokinase acted simultaneously, they

would constitute a substrate cycle in which 1,6-bisphosphate and

fructose-6-phosphate became interconverted with net consumption of ATP:

Fructose-1,6-BP  H2O⎯⎯→ fructose-6-P  Pi Fructose-6-P  ATP ⎯⎯→ fructose-1,6-BP  ADP

Because substrate cycles such as this appear to operate with no net benefit to the cell, they were once regarded as metabolic quirks and were referred to as futile cycles More recently, substrate cycles have been recognized as important devices for controlling metabolite concentrations

The three steps in glycolysis and gluconeogenesis that differ constitute three such substrate cycles, each with its own particular metabolic raison d’être Consider, for example, the regulation of the fructose-1,6-BP–fructose-6-P cycle by fructose-2,6-bisphosphate As already noted, fructose-1,6-bisphosphatase is subject to allosteric

in-20

Fructose-1,6-bisphosphatase activity, units/mg protein

(a)

15

10

5

0 0 Fructose-1,6-bisphosphate,

50 100 200

Fructose-1,6-bisphosphatase activity, units/mg protein

(b)

15

10

5

0 0 Fructose-1,6-bisphosphate,

50 100 200

0 1

5

25

0 0.2 1

2.5 5

100

(c)

75

50

25

0 0 Fructose-2,6-bisphosphate,

0

10

25

FIGURE 22.9 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and

(b) presence of 25 mM AMP In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate)

concentration Concentrations of fructose-2,6-bisphosphate (in mM) are indicated above each curve (c) The effect

of AMP (0, 10, and 25 mM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate Activity was measured in the presence of 10 mM fructose-1,6-bisphosphate.(Adapted from Van Schaftingen, E., and Hers, H-G.,

1981 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate Proceedings of the National Academy of Science, U.S.A.

78:2861–2863.)

Pi

H2O F2,6-BP

F2,6-BPase

F-6-P F-6-P

ADP F2,6-BP

cAMP-dep PK

ADP

PFK-1 PFK-2

F2,6-BPase PFK-2

F1,6-BPase

ATP ATP

+ –

–

FIGURE 22.10 (a) Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same bifunctional

enzyme (b) The structure of PFK-2/F-2,6-BPase from rat liver PFK-2 activity resides in the N-terminal portion of the

protein (left), and the C-terminal domain (right) contains F-2,6-BPase activity.The PFK-2 domain has a bound ATP

analog; the F2, 6-Pase has two phosphates bound (pdb id  1BIF).

stimu-lates bisphosphatase activity, but fructose-2,6-bisphosphate is a potent allosteric

in-hibitor AMP also inhibits the bisphosphatase; the inhibition by... be under subsub-strate-level control by

glucose-6-phosphate

Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis It al-losterically inhibits pyruvate... class="text_page_counter">Trang 9

bisphosphate is a potent stimulator of phosphofructokinase (see Chapter 18)

Cog-nizant of the reciprocal nature