Pentose Phosphate Pathway

The Pentose Phosphate Pathway

A prerequisite for this article is knowledge of the glycolytic pathway. Refer to this blogpost for a detailed account of glycolysis.

The pentose phosphate pathway (also known as the hexose monophosphate (HMP) shunt pathway) is closely related to lipid biosynthesis, nucleotide biosynthesis, glycolysis and gluconeogenesis. To properly understand how these pathways are related, it is important to first understand what the pentose phosphate pathway does.

Goals of the pathway

The goals of the pentose phosphate pathway pathway are three fold:

• To synthesize NADPH
• To synthesize Ribose-5-Phosphate (R5P)
• To provide building blocks for amino acids

ATP is the cell’s “energy currency”; its exergonic hydrolysis is coupled to many otherwise endergonic cell functions. Cells have a second currency, reducing power. - Donald Voet and Judith G Voet

NADPH is involved in utilizing the free energy of metabolite oxidation for otherwise endergonic reductive biosynthesis. Thus, it functions as the reducing power of the cell. The primary utility of this reducing power lies in the biosynthesis of lipids. Note that NADH and NADPH are not biologically interchangeable. Their biochemical roles vary and their enzymes exhibit high degrees of specificity.

Ribose-5-phosphate is required for nucleotide biosynthesis. The ribose-5-phosphate formed via the pentose phosphate pathway is converted into phosphoribosyl pyrophosphate (PRPP) in the fist step of nucleotide biosynthesis.

Phases of the pentose phosphate pathway

The pentose phosphate pathway runs in two modes: the oxidative mode and the non-oxidative. Oxidation of pathway intermediates takes place in the oxidative mode. These oxidation reactions are coupled with the reduction of NADP$^+$ to NADPH.

The intermediates resulting form the completion of the oxidative mode are pentoses (ribose-5-phosphoate, ribulose-5-phosphate and xylulose-5-phosphate). The carbon skeleton of these intermediates is rearranged such that fructose-6-phosphate and glyceraldehyde-3-phosphate are formed from them. The steps that rearrange the carbon skeleton or the “carbon-scrambling” steps are reversible in nature. Thus, pentoses can be synthesized from fructose-6-phosphate and glyceraldehyde-3-phosphate. The carbon scrambling phase constitutes the non-oxidative mode of the pentose phosphate pathway.

When the metabolic need for NADPH is greater than that for pentoses, the pathway operates in the oxidative mode (above). The pentoses undergo carbon scrambling (to form fructose-6-phosphate and glyceraldehyde-3-phosphate) and are fed back into the glycolytic pathway. If the cellular need for NADPH persists, then fructose-6-phosphate and glyceraldehyde-3-phosphate can be converted back to glucose-6-phosphate via gluconeogenesis and can re-enter the oxidative mode of the pentose phosphate pathway. Thus, a continuous cycle of NADPH generation is formed. The yellow curved arrow in the figure above represents this cycle.

When the metabolic requirement for pentoses is greater than that for NADPH, the pentose phosphate pathway operates in the non-oxidative mode. Here, fructose-6-phosphate and glyceraldehyde-3-phosphate enter the pathway, undergo carbon scrambling and produce pentoses that are then channelized off to nucleotide synthesis or other pathways that require them.

Modes of operation of the pentose phosphate pathway:
a) Oxidative mode: Requirement of NADPH
b) Non-oxidative mode: Requirement of pentoses

Stages in the pathway

The stages in the pathway:
1)Production of 2 NADPH
2)Interconversion of pentoses
3)Carbon scrambling of the pentoses into glycolytic intermediates (fructose-6-phosphate 
   and glyceraldehyde-3-phosphate)

The pathway

All reactions of the carbon scrambling phase are reversible. The product molecules are highlighted with yellow.

The above pentose phosphate pathway can be summarized in the following reaction:
3 glucose-6-phosphate + 2 NADP^+ $\rightarrow$ 2 fructose-6-phosphate + glyceraldehyde-3-phophate + 2 NADPH

Production of 2 NADPH molecules

1) Glucose-6-phosphate is converted to phosphogluconolactone
Enzyme: Glucose-6-phosphate dehydrogenase (G6PD)

In the fist step of the pathway, the substrate glucose-6-phosphate transfers a hydride ($H^-$) from its first carbon to NADP$^+$ to form the first NADPH molecule. The consequent oxidation of glucose-6-phosphate results in the formation of a cyclic ester, 6-phosphogluconolactone.

2) Phosphogluconolactone is hydrolyzed
Enzyme: Gluconolactone hydrolase

Hydrolysis of 6-phosphogluconolactone results in the formation of a $-CO^-_2$ group at carbon $1$. The compound formed, 6-phosphogluconate is the anion of a $\beta$-hydroxy acid.

3) 6-Phosphogluconate is converted into ribulose-5-phosphate (Ru5P)
Enzyme: Gluconate dehydrogenase

The $\beta$-hydroxy acid, 6-phosphogluconate undergoes dehydrogenation to form a beta ketoacid. The significance of this step is two fold:

• Firstly, a $H^-$ ion is removed and NADPH is simultaneously formed.
• Secondly, a $\beta$-ketoacid formed which readily undergoes decarboxylation

Thus, by the end of this step, the first 5 carbon intermediate, ribulose-5-phosphate (Ru5P) is formed. The mechanism of this reaction is similar to that of isocitrate dehydrogenase of the citric acid cycle. An intermediate $\beta$-ketoacid (susceptible to decarboxylation) is formed in both the reactions.

Interconversion of pentoses

4) Ribulose-5-phosphate is isomerized to ribose-5-phosphate
Enzyme: Ribose-5-phosphate ketoisomerase

The formation of ribose-5-phosphate occurs in a single isomerization reaction that proceeds via an enediolate intermediate. The ribose-5-phosphate thus formed is channelized for nucleotide synthesis, the first step of which involves the formation of phosphoribosyl pyrophosphate (PRPP).

5) Ribulose-5-phosphate is epimerized into xylulose-5-phosphate
Enzyme: Ribulose-5-phosphate 3 epimerase

Ribulose-5-phospate is epimerized to xylulose-5-phosphate. This reaction occurs at a faster rate than the previous reaction. Thus, for every three molecules of ribulose-5-phosphate, two molecules of xylulose-5-phosphate and only one molecule of ribose-5-phosphate are formed.

Carbon scrambling: rearrangement of pentoses to glycolytic intermediates

6) Xylulose-5-phosphate and ribose-5-phosphate combine
Enzyme: Transketolase

The keto group of one of the xylulose-5-phosphate molecules is transferred onto the carbonyl carbon of the ribose-5-phosphate to give rise to a seven carbon compound sedoheptulose-7-phosphate and a three carbon compound glyceraldehyde-3-phosphate. Transketolase uses TPP (thiamine pyrophosphate) as a cofactor to stabilize the carbanion formed during the transfer of the keto group.

7) Carbon skeleton is rearranged into fructose-6-phosphate and erythrose-4-phosphate
Enzyme: Transaldolase

This reaction is reversible under physiological conditions.

The mechanism of the enzyme transaldolase is similar to that of the class $\rm I$ aldolase used in glycolysis to cleave fructose-1,6-bisphosphate into glyceraldehyde-3-phophate and dihydroxyacetone phosphate. Transaldolase performs an aldol cleavage between the $\alpha$ and $\beta$ carbons of sedoheptulose-7-phosphate resulting in erythrose-4-phosphate (figure above). The cleaved off three carbon group is then transferred onto glyceraldehyde-3-phosphate to give fructose-6-phosphate. Thus, the first fructose molecule is formed. This molecule enters glycolysis or gluconeogenesis.

8) Glyceraldehyde-3-phosphate and another fructose-6-phosphate are formed
Enzyme: Transketolase

The keto group of the second molecule of xylulose-4-phosphate (formed in step 5) is cleaved off resulting in glyceraldehyde-3-phosphate. This keto group is added onto erythrose-4-phosphate to form the second molecule of fructose-6-phosphate.

The differences between transketolase and transaldolase are explained in greater detail in this stack exchange answer.

Regulation of the pathway

The flux through the oxidative pentose phosphate pathway is controlled by the rate of the enzyme glucose-6-phosphate dehydrogenase (G6PD of reaction 1). This reaction is irreversible due to its highly exothermic nature.
$\Delta G _{reaction \text{ }1}= -17.6 \text{ }kJ/mol$

The concentration of NADP$^+$ regulates G6PD activity. As the cell consumes NADPH, the level of NADP$^+$ rises, this increases the rate of G6PD.

G6PD mutants and deficiency

Deficiency of glucose-6-phosphate dehydrogenase is the most common human enzymopathy. This condition results in the deficiency of NADPH which is required for several reductive processes in addition to biosynthesis. For example, glutathione reductase, an enzyme that eliminates peroxides in red blood cells requires NADPH as a cofactor (above figure). Peroxides are harmful because they react with double bonds in the fatty acid residues of the erythrocyte cell membrane to form organic hydroperoxides. These, in turn, react to cleave fatty acid $C-C$ bonds, thereby damaging the membrane. Thus, buildup of peroxides can lead to premature cell lysis. This results in hemolytic anemia. The patient presents with jaundice and an enlarged spleen (among other symptoms) as a direct consequence of increased hemolysis.

A steady supply of NADPH is vital for erythrocyte integrity. Thus, G6PD deficiency causes red blood cells to break down in response to certain medication, infections or other stresses that stimulate peroxide formation, causing hemolytic anemia.

In vitro studies indicate that erythrocytes with G6PD deficiency are less suitable hosts for plasmodia than normal cells. This is presumably because the erythrocyte is lysed before the parasite has had a chance to mature. Thus, like the sickle-cell trait, a defective G6PD confers a selective advantage on individuals living where malaria is endemic.

Sources

• Thumbnail: RBC lysis due to G6PD deficiency.
• MITOCW lecture video
• Harper’s Illustrated Biochemistry 31st Edition
• Biochemistry by Donald Voet and Judith G. Voet
• Khan Academy blogpost