Pentose Phosphate Pathway

Three main functions:

1) Supply the cell with NADPH in order to:

a) provide reducing power for biosynthetic reactions.

b) serve as a biochemical reductant (e.g., maintain glutathione levels).

c) be utilized by the cytochrome P450 monooxygenase system.

d) as the electron source for reduction of ribo- to deoxyribonucleotides

for DNA synthesis.

2) Convert hexoses into pentoses (which are essential components of

ATP, CoA, NADP+, FAD, RNA, and DNA).

3) Enable the complete oxidative degradation of pentoses by

converting them into hexoses and trioses which can then enter

the glycolytic pathway.

The Roles of NADH and NADPH in Metabolism

There is a fundamental distinction between NADH and NADPH in

most biochemical reactions.

NADH is oxidized by the electron transport chain to generate ATP.

In contrast, NADPH functions as an electron donor (i.e., a hydride

ion donor) in biosynthetic reactions.

Recall that in the oxidation of a substrate, the nicotinamide ring of

NADP+ accepts a hydrogen ion and two electrons, which are

equivalent to a hydride ion.

Stages of the Pentose Phosphate Pathway

Stage 1: consists of the oxidative portion of the pathway in which

two oxidative reactions provide NADPH and a hexose is decarboxylated

to a pentose.

Stage 2: consists of two reversible isomerization reactions.

Stage 3: consists of the nonoxidative portion of the pathway in which

via a series of interconversions of three-, four-, five-, six-, and

seven-carbon sugars, excess pentoses are converted to hexoses

and trioses which can enter the glycolytic pathway.

Stage 1: Three reactions constitute this stage, two of which are

oxidative and generate NADPH.

Stage 1 is linked to biosynthetic reactions since NADPH and a

pentose are produced.

The reactions of stage 1 can be summarized as follows:

Glucose 6-phosphate + 2 NADP+ + H2O

ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2

Thus two of the three functions of the pentose phosphate pathway are

accomplished: generation of NADPH and conversion of a hexose to a

pentose.

Glucose 6-phosphate

dehydrogenaseLactonase

6-phosphogluconate

dehydrogenase1

1

3

oxidative

decarboxylation

Stage 2 consists of two reversible isomerization reactions which convert

ribulose 5-phosphate into either ribose 5-phosphate or xylulose

5-phosphate.

Both are substrates for the Stage 3 reactions.

Ribulose 5-phosphate can also be isomerized to xylulose 5-phosphate

via the enzyme phosphopentose epimerase.

Phosphopentose isomerase

ketose

aldose

The Stage 1 + Stage 2 reactions yield 2 NADPH and 1 ribose

5-phosphate for each glucose 6-phosphate oxidized.

However, cells often need NADPH reducing power more than they

need ribose 5-phosphate for nucleotide biosynthesis.

In these cases, ribose 5-phosphate is further converted into

glyceraldehyde 3-phosphate and fructose 6-phosphate by the

enzymes transketolase and transaldolase.

These enzymes created a reversible link between the pentose

phosphate pathway and glycolysis.

Reactions of Stage 3

Stage 3 consists of non-oxidative reactions which link the pentose

phosphate pathway with glycolysis.

This stage allows:

1) excess pentoses to be converted to hexoses and trioses which can

then enter glycolysis; and

2) hexoses to be converted to pentoses, thereby allowing pentose

production without concomitant production of NADPH.

Two enzymes – transketolase and transaldolase – catalyze a series of

three reactions which convert 3 pentoses into 2 hexoses and 1 triose.

These reactions involve interconversions of 3, 4, 5, 6, and

7-carbon sugars.

Transketolase transfers a 2-carbon fragment.

Transaldolase transfers a 3-carbon fragment.

C5 + C5 C3 + C7

C7 + C3 C4 + C6

C5 + C4 C3 + C6

Net: 3 C5 2 C6 + 1 C3

transketolase

transaldolase

transketolase

Glyceraldehyde

3-phosphate

Fructose

6-phosphate

Reaction 1

Two pentoses are required: ribose 5-phosphate and xylulose

5-phosphate.

A 2 carbon fragment is transferred from the ketose to the aldose.

Catalyzed by transketolase.

Ketose

Aldose

C5 C5 C3 C7

Transketolase contains tightly bound TPP as its prosthetic group.

Wernicke Kosakoff Syndrome is an autosomal recessive disorder caused by

an alteration in transketolase which reduces its affinity for TPP.

Symptoms only develop if individual suffers from a moderate thiamine deficiency.

Reaction 2

The products of Reaction 1 (i.e., glyceraldehyde 3-phosphate and

sedoheptulose 7-phosphate) are the substrates for Reaction 2.

A 3-carbon unit is transferred from the ketose to the aldose by the

enzyme transaldolase.

C7 C3 C4 C6

Transketolase is also utilized for the third reaction.

A 2-carbon unit is transferred from xylulose 5-phosphate (a ketose) to

erythrose 4-phosphate (an aldose).

Note: the products of this reaction, glyceraldehyde 3-phosphate and

fructose 6-phosphate, are both intermediates of the glycolytic

pathway.

C5 C4 C3 C6

The sum of the Stage 3 reactions is:

2 Xylulose 5-phosphate + ribose 5-phosphate

2 fructose 6-phosphate + glyceraldehyde 3-phosphate

If we include the Stage 2 isomerization reactions, the net reaction is:

3 Ribose 5-phosphate

2 fructose 6-phosphate + glyceraldehyde 3-phosphate

The important point is that excess ribose 5-phosphate formed by the

pentose phosphate pathway can be completely converted into

glycolytic intermediates.

The Rate Limiting Step of the Pentose Phosphate Pathway

The first reaction in the oxidative branch of the pentose phosphate

pathway catalyzed by glucose 6-phosphate dehydrogenase, is the

rate limiting step under physiological conditions.

NADPH is a potent competitive inhibitor of the enzyme. Thus, the ratio

of NADP+/NADPH regulates the pathway.

As the NADP+ level rises, the flux thru the pathway increases.

Note: The nonoxidative branch of the pathway is regulated primarily by

substrate availability.

The Flow of Glucose 6-phosphate Depends on

Physiological Need

The flow of glucose 6-phosphate depends on cellular need for NADPH,

ribose 5-phosphate, and ATP. The Pentose Phosphate Pathway can

operate in 4 different modes.

Mode 1: Much more ribose 5-phosphate than NADPH is required.

Also need:

phosphopentose isomerase

phosphopentose epimerase

For example, rapidly dividing

cells need nucleotide precursors

for DNA synthesis more than

they need NADPH.

Glycolysis 2

1

3

transketolase

transaldolase

Bypass Stage 1, feed into

glycolysis instead.

Use glycolysis +

Stage 2 and Stage 3

reactions.

Mode 2: The needs for NADPH and ribose 5-phosphate are balanced.

Oxidative Branch – Stage 1

Phosphopentose

isomerase

The stoichiometry of mode 2 is:

Glucose 6-phosphate + 2 NADP+ + H2O

ribose 5-phosphate + 2 NADPH + 2 H+ + CO2

Mode 3: Much more NADPH than ribose 5-phosphate is required;

glucose 6-phosphate is completely oxidized to CO2.

Three sets of reactions are required under these conditions.

This situation typically occurs

in adipose tissue where a high

level of NADPH is required for

fatty acid biosynthesis.

The sum of these reactions is:

Glucose 6-phosphate + 12 NADP+ + 7 H2O

6 CO2 + 12 NADPH + 12 H+ + Pi

Oxidative Branch

Phosphopentose

isomerase

transketolase

transaldolase

Gluconeogensis

(1)

(2)

(2)

(3)

(2)

Regenerate glucose 6-phosphate

from the pentose.

Mode 4: Much more NADPH than ribose 5-phosphate is required;

glucose 6-phosphate is converted into pyruvate.

ATP, NADH, and NADPH

are generated.

Five of the six carbons of

glucose 6-phosphate emerge

as pyruvate.

The stoichiometry of mode 4 is:

3 Glucose 6-phosphate + 6 NADP+ + 5 NAD+ + 5 Pi + 8 ADP

5 pyruvate + 3 CO2 + 6 NADPH + 5 NADH + 8 ATP + 2 H2O + 8 H+

Oxidative Pathway

transketolase

transaldolaseGlycolysis

(1)

(2)(3)

ATP or Biosynthetic

precursors

Phosphopentose

isomerase

The Percentage of Glucose Metabolized by the Pentose

Phosphate Pathway Varies for Different Tissues

Since, a main purpose of this pathway is to supply NADPH for reductive

syntheses, it is prominent in tissues that actively carry out the reductive

synthesis of fatty acids and/or steroids from acetyl CoA.

Liver: 5-10% of glucose is metabolized by the pentose

phosphate pathway.

Adipose Tissue: 30 – 50%

Erythrocytes: 10% (need NADPH to maintain reduced glutathione)

Thyroid gland, kidney, and brain: 3 – 5%

Muscle: activity is extremely low.

A radioisotopic approach can be used to assess the fraction of glucose

metabolized by the pentose phosphate pathway vs. the sum of

glycolysis, PDH, + the citric acid cycle in a given tissue.

Principle: Only the C-1 position of glucose is decarboxylated by the

pentose phosphate pathway (at the 6-phosphogluconate dehydrogenase

step).

In contrast, the C-1 and C-6 positions are decarboxylated equally when

glucose is metabolized by the glycolytic pathway, PDH, and the citric

acid cycle.

This is because in these pathways, the C-1 and C-6 from glucose end up

as C-3 in pyruvate following the triose phosphate isomerase step. Thus,

subsequent oxidation by the citric acid cycle liberates 14CO2 equally from

the original C-1 and C-6 of glucose.

Thus, 2 samples of a given tissue are prepared:

One is incubated with glucose labeled with 14C at position C-1,

whereas the other sample is incubated with glucose labeled at

position C-6. The amount of radioactive 14CO2 produced by the 2

samples is then compared.

For example, in liver:

14CO2 liberated by the sample incubated in glucose-1-14C is produced

by both the citrate acid cycle and the pentose phosphate pathway;

However, less 14CO2 is liberated by the sample incubated in

glucose-6-14C because the citric acid cycle is the only source of14CO2.

By comparing the amount of 14CO2 released with different tissues

that are labeled in the C-1 versus C-6 positions, one can determine

the proportion of glucose metabolized by one pathway versus another.

Role of Glucose 6-phosphate Dehydrogenase in the

Red Blood Cell

In the RBC glucose serves as the primary energy source. RBC’s lack

mitochondria and thus lack the enzymes of the citric acid cycle.

Therefore, glucose is metabolized exclusively by the glycolytic

pathway (90%) and the pentose phosphate pathway (10%).

The most important function of the pentose phosphate pathway in the

RBC is to maintain the tripeptide glutathione in a reduced state.

Oxidized glutathione is reduced by the enzyme glutathione reductasein a reaction which utilizes NADPH:

Functions of Reduced Glutathione

1) To serve as a sulfhydryl buffer that maintains the cysteine residues

of hemoglobin and other RBC proteins in the reduced state.

2) To maintain the iron in hemoglobin in the ferrous (i.e., reduced)

form (Fe++).

3) To detoxify by reacting with hydrogen peroxides and organic

peroxides. This reaction is catalyzed by glutathione peroxidase.

H2O2 + 2 GSH 2 H2O + GSSG

Thus reduced glutathione is essential for maintaining the normal

structure of RBCs. Cells with a lowered level of this compound

are more susceptible to hemolysis.

Certain drugs act as oxidants formation of toxic peroxides

oxidation of proteins distortion of the surface of RBCs in the

absence of glutathione, making them more susceptible to destruction.

Glucose 6-phosphate Dehydrogenase Deficiency

•Glucose 6-phosphate dehydrogenase (G6PD) deficiency is an inherited

disease characterized by hemolytic anemia caused by an inability to

detoxify oxidizing agents.

•Most common disease-producing enzyme abnormality in humans.

•Caused by a family of over 400 point mutations in the enzyme.

•Only certain mutations cause clinical symptoms.

•Most affected individuals have no symptoms until they are exposed to

certain drugs which act as oxidizing agents.

•Drug exposure can induce a hemolytic episode which in some cases

can be fatal.

•Life-span of many individuals with G6PD deficiency is shortened due to

complications arising from chronic hemolysis.

Genetic Variants of Glucose 6-phosphate Dehydrogenase

More than 400 putative variants of the G6PD have been described.

Among the properties by which the variants can be distinguished

are the following:

1) enzyme activity

2) kinetic properties

3) electrophoretic mobility

4) substrate specificity

5) enzyme stability

6) pH optimum

The severity of the disease typically correlates with the amount of

residual enzyme activity in the patient’s RBCs.

•G6PD A- is the prototype of the moderate (class III) form of the disease.

•The A- variant occurs with high frequency amongst the African-American

population (gene frequency of the A- allele ~ 11%) and causes a

susceptibility to drug-induced hemolytic anemia.

•The enzyme encoded by the A- allele is kinetically normal, but displays

a substantially reduced half-life (i.e., 13 days versus 62 days).

Consequence: about 3 days after an oxidizing drug is administered to an

A- individual, there is a pronounced hemolytic episode. Older RBCs

which have little functional enzyme and hence are deficient in NADPH

and reduced glutathione are destroyed.

After ~ 1 week recovery begins since most of the remaining (i.e., younger

RBCs), as well as newly produced RBCs, have relatively normal enzyme

levels.

Provides protection against the drug-induced hemolysis.

Glucose 6-phosphate Dehydrogenase Deficiency

•The high frequency of the A- variant suggests that the deficiency may

be advantageous under certain environmental conditions.

•In fact it confers partial protection against malaria (since the causative

parasite requires reduced glutathione and pentose phosphate pathway

products).

•Illustrates the interplay between heredity and environment.

The G6PD Mediterranean variant is the prototype of a more severe

(class I) deficiency.

•Enzyme shows normal stability, but barely detectable activity in RBCs.

•A broader spectrum of drugs causes hemolysis and the hemolysis

is not as self limiting as in individuals with the A- variant.

Molecular Biology of Glucose 6-phosphate Dehydrogenase

•The cloning of the G6PD gene has enabled identification of mutations

that cause G6PD deficiency. All are point mutations in the coding

region of the gene.

•Mutations causing nonspherocytic hemolytic anemia: cluster near the

the NADP+ binding site.

•Mutations causing milder forms of the disease cluster near the glucose

6-phosphate binding site.

Organ Integration of Carbohydrate Metabolism

Liver: essential for providing fuel (i.e., glucose) to the brain, muscle,

and other peripheral organs.

The liver extracts Glucose from the blood.

Glycogen

Release glucose

when needed.

Pentose Phosphate

Pathway

NADPH for biosynthesis;

pentoses, hexoses.

Glycolysis

Mainly as a source

of biosynthetic

intermediates; also

to make some ATP

Note:

1) If there is insufficient glucose in the blood, the liver will break down its

glycogen and/or synthesize glucose via gluconeogenesis to increase

blood glucose supplied to other tissues.

2) Liver mainly uses α-keto acids derived from amino acid catabolism to supply

its own ATP needs.

Brain: Glucose is virtually the sole fuel source for the human brain,

except during times of starvation. In the resting state, the brain

accounts for 60% of glucose utilization.

•Glucose is obtained either from the diet or the liver.

•Brain does not contain significant stores of glycogen.

•Brain does not carry out gluconeogenesis since it has no glucose

6-phosphatase.

Muscle: In contrast to brain, muscle has a large store of glycogen

(i.e., ~ 75% of total body glycogen).

•Its major fuels are glucose, fatty acids, and ketone bodies.

•Like brain, muscle lacks glucose 6-phosphatase, and so it does not

carry out gluconeogenesis or export glucose.

•Instead muscle retains glucose, its preferred fuel, for bursts of activity.

Metabolic Interchanges Between Muscle and Liver

•During active contraction, glycolysis >> citric acid cycle. Therefore,

pyruvate is reduced to lactate which then flows to liver where it is

converted into glucose (Cori Cycle).

•Muscle also produces much alanine (by transamination of pyruvate).

This alanine can also be converted into glucose by the liver.

Adipose Tissue: Triacylglycerols stored in adipose tissue provide

an enormous reservoir of metabolic fuel.

•A principal function of this tissue is to synthesize triacylglycerols from

fatty acyl CoA derivatives and glycerol 3-phosphate.

•Thus, adipose cells need to metabolize glucose via glycolysis in order

to provide sufficient glycerol 3-phosphate (which originates from

dihydroxyacetone phosphate) for triacylglycerol synthesis.

•Also, adipose cells need to carry out some pentose phosphate pathway

in order to supply sufficient NADPH for synthesis.

Metabolic Relationships in the Well-Fed State