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Q.1- What is HMP pathway? Explain in detail the steps of this pathway.

Answer- The pentose phosphate pathway (also called Phospho gluconate pathway or hexose monophosphate shunt [HMP shunt]) is an alternative route for the metabolism of glucose. It does not lead to formation of ATP but has two major functions: (1) The formation of NADPH for synthesis of fatty acids and steroids, and (2) the synthesis of ribose for nucleotide and nucleic acid formation.

Overview of HMP pathway

The pentose phosphate pathway meets the need of all organisms for a source of NADPH to use in reductive biosynthesis .This pathway consists of two phases: the oxidative generation of NADPH and the nonoxidative interconversion of sugars (Figure 1). In the oxidative phase, NADPH is generated when glucose 6-phosphate is oxidized to ribose 5-phosphate. This five-carbon sugar and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A.

In the nonoxidative phase, the pathway catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of nonoxidative reactions that can result in the synthesis of five-carbon sugars for nucleotide biosynthesis or the conversion of excess five-carbon sugars into intermediates of the glycolytic pathway. All these reactions take place in the cytosol.

Figure 1- showing an overview of HMP pathway- GAP (Glyceraldehyde-3-phosphate)

The pentose phosphate pathway is a more complex pathway than glycolysis (Figure 1). Six molecules of glucose 6-phosphate give rise to six molecules of CO2 and six five-carbon sugars, these are rearranged to regenerate 4 molecules of glucose 6-phosphate and 2 molecule of the glycolytic intermediate, glyceraldehyde 3-phosphate. Since two molecules of glyceraldehyde 3-phosphate can regenerate glucose 6-phosphate, hence 5 molecules glucose 6-phosphate are regenerated. The generation of 6 molecules of CO2 in  the pathway can account for the complete oxidation of  one molecule of glucose. 

Reactions of HMP pathway

The sequence of reactions of the pathway may be divided into two phases: an oxidative nonreversible phase and a nonoxidative reversible phase.

1) Oxidative Phase-Unlike glycolysis, oxidation is achieved by dehydrogenation using NADP+, not NAD+, as the hydrogen acceptor. The oxidative phase of the pentose phosphate pathway starts with the dehydrogenation of glucose 6-phosphate at carbon 1, a reaction catalyzed by glucose 6-phosphate dehydrogenase (Figure 2). This enzyme is highly specific for NADP+; the Km for NAD+ is about a thousand times as great as that for NADP+. The product is 6-phosphoglucono-δ-lactone, which is an intramolecular ester between the C-1 carboxyl group and the C-5 hydroxyl group. The next step is the hydrolysis of 6-phosphoglucono- δ -lactone,  by a specific lactonase(Hydrolase) to give 6-phosphogluconate. This six-carbon sugar is then oxidatively decarboxylated by 6-phosphogluconate dehydrogenase to yield Ribulose 5-phosphate. NADP+ is again the electron acceptor. The final step in the synthesis of ribose 5-phosphate is the isomerization of Ribulose 5-phosphate by phosphopentose Isomerase (see Figure)

Figure 2- showing the reactions of oxidative phase of HMP pathway

The Nonoxidative Phase

Ribulose 5-phosphate is the substrate for two enzymes. Ribulose 5-phosphate 3-epimerase(Phosphopentose epimerase) alters the configuration about carbon 3, forming the epimer Xylulose 5-phosphate, also a ketopentose. (Figure 3)

Ribose 5-phosphate keto Isomerase (Phosphopentose isomerase) converts Ribulose 5-phosphate to the corresponding aldopentose, ribose 5-phosphate, which is the precursor of the ribose required for nucleotide and nucleic acid synthesis.(Figure 3)

Figure 3- showing interconversion of pentoses

Transketolase transfers the two-carbon unit comprising carbons 1 and 2 of a ketose onto the aldehyde carbon of an aldose sugar. It therefore effects the conversion of a ketose sugar into an aldose with two carbons less and an aldose sugar into a ketose with two carbons more. The reaction requires Mg2+ and thiamin diphosphate (vitamin B1) as coenzyme. The two-carbon moiety transferred is probably glycoaldehyde bound to thiamin diphosphate. Thus, transketolase catalyzes the transfer of the two-carbon unit from Xylulose 5-phosphate to ribose 5-phosphate, producing the seven-carbon ketose sedoheptulose 7-phosphate and the aldose glyceraldehyde 3-phosphate. These two products then undergo transaldolation.(Figure 4)

Figure 4- showing the reaction catalyzed by transketolase

Transaldolase catalyzes the transfer of a three-carbon Dihydroxyacetone moiety (carbons 1–3) from the ketose sedoheptulose 7-phosphate onto the aldose glyceraldehyde 3-phosphate to form the ketose fructose 6-phosphate and the four-carbon aldose erythrose 4-phosphate. (Figure-5)

Figure 5- showing the reaction catalyzed by transaldolase

In a further reaction catalyzed by transketolase, Xylulose 5-phosphate serves as a donor of glycoaldehyde. In this case erythrose 4-phosphate is the acceptor, and the products of the reaction are fructose 6-phosphate and glyceraldehyde 3-phosphate. (Figure 6)

Figure 6- showing the rearrangement of sugars to form glycolytic intermediated catalyzed by transketolase

In order to oxidize glucose completely to CO2 via the pentose phosphate pathway, there must be enzymes present in the tissue to convert glyceraldehyde 3-phosphate to glucose 6-phosphate. This involves reversal of glycolysis and the gluconeogenic enzyme fructose 1,6-bisphosphatase. In tissues that lack this enzyme, glyceraldehyde 3-phosphate follows the normal pathway of glycolysis to pyruvate.

The sum of these reactions is

Xylulose 5-phosphate can be formed from ribose 5-phosphate by the sequential action of phosphopentose isomerase and phosphopentose Epimerase, and so the net reaction starting from ribose 5-phosphate is

Thus, excess ribose 5-phosphate formed by the pentose phosphate pathway can be completely converted into glycolytic intermediates. Moreover, any ribose ingested in the diet can be processed into glycolytic intermediates by this pathway.

It is evident that the carbon skeletons of sugars can be extensively rearranged to meet physiologic needs.

Q.2- Enlist the important differences between Glycolysis and HMP pathway.

 Answer-

Characteristics Glycolysis HMP pathway
Occurrence All cells of the body Active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary glands.
Glucose Oxidation(Coenzyme) Oxidation is achieved by dehydrogenation using NAD+ as the hydrogen acceptor. Oxidation is achieved by dehydrogenation using NADP+ as the hydrogen acceptor.
CO2 production CO2 is not produced CO2 is produced
Pentose production Pentoses are not produced Pentoses are produced
Intermediates Can be in the bisphosphate form- such as Fr 1,6 bisphosphate , 1,3 bisphosphoglycerate or 2,3 bisphosphoglycerate etc. Never in bisphosphate form. Always in mono phosphate form that is why called Hexose mono phosphate pathway.
Energy ATP is utilized as well as produced. ATP is a major product of glycolysis ATP is neither utilized nor produced.Glycolytic intermediates may enter glycolytic pathway to produce energy
 Biological Significance Energy production both in aerobic and anaerobic conditions NADPH is required for reductive biosynthesis and pentoses are required forsynthesis of coenzymes and nucleotides.
Clinical Significance Hemolytic anemia in Pyruvate kinase and Hexokinase deficiency Hemolytic anemia in Glucose-6-P dehydrogenase deficiency

 

Q.3- The pentose phosphate pathway is active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary gland. Why is the pathway less active in non-lactating mammary gland and skeletal muscle? 

Answer- HMP pathway is highly active in rapidly dividing cells and in tissues where there is a great requirement of NADPH. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione

In liver NADPH is required for the synthesis of fatty acids, sterols, cholesterol and for the activity of Glutamate dehydrogenase,

In adipose tissue and in lactating mammary glands, NADPH is required for fatty acids synthesis.

It is required for the synthesis of steroid hormones in adrenal cortex

Similarly it is required for the synthesis of hormones in testes, ovaries and in thyroid gland.

In lens and in Erythrocytes NADPH is required for reduction of Glutathione which is an essential component of Glutathione peroxidase, meant for maintaining the integrity of these tissues.

In skeletal, muscle, this pathway is less active. Muscle tissue contains very small amount of dehydrogenases but skeletal muscle is capable of synthesizing ribose. Probably, this is accomplished by reversal of non oxidative phase of shunt pathway utilizing fructose-6-P and Glyceraldhyde-3-P under the activities of Transketolase and Transaldolase enzymes

Q.4- Discuss the regulation of HMP pathway.  Under what conditions is this pathway stimulated or inhibited?

Answer- Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway.  Following factors affect the activity of this enzyme and thus influence the rate of this pathway-

 I) Availability of Substrate- This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH.  The inhibitory effect of low levels of NADP+ is exacerbated by the fact that NADPH competes with NADP+ in binding to the enzyme. The low ratio of NADP+ to NADPH stimulates the enzyme while the high ratio inhibits the enzyme. The marked effect of the NADP+ level on the rate of the oxidative phase ensures that NADPH generation is tightly coupled to its utilization in reductive biosyntheses. This explains the higher rate of activity of HMP pathway in tissues involved in reductive biosynthesis.

2) Induction and repression- The synthesis of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase may also be induced by insulin in the fed state, when lipogenesis increases.  Excessive carbohydrate ingestion thereby leads to more NADPH generation.

The synthesis of these enzymes is decreased in the fasting state.

The non oxidative phase of HMP pathway accomplishes conversion of the 5-C ribulose-5-phosphate to the 5-C product ribose-5-phosphate, or to the 3-C glyceraldehyde-3-phosphate and the 6-C fructose-6-phosphate. This phase is regulated by the flow of substrates.

 

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