Pentose Phosphate Pathway PDF

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Pozzi BICH411 Lecture Supplement Notes

Pentose Phosphate Pathway Consider glucose metabolism, we know that glucose is phosphorylated forming glucose 6-phosphate so that it is trapped inside the cell for cellular use. We have looked at the use of glucose for glycolysis, the fates of pyruvate depending on oxygen concentration. Under aerobic conditions, pyruvate is feed through the pyruvate dehydrogenase complex into the citric acid cycle in the mitochondria, whereas under anaerobic conditions, pyruvate is converted to lactate. Lactate must be dealt with and the Cori cycle is what allows for transport of lactate from muscles to the liver for gluconeogenesis so that glucose and be generated for transport back to the tissues. However, in looking at these pathways, we see that glucose oxidation serves to facilitate synthesis of ATP (goal of both aerobic and anaerobic fates of pyruvate ultimately produce ATP for the cell). But what do we know to be the importance of catabolic contributions to the cell? ATP production, and also NADPH production – as both of these are necessary for biosynthetic pathways or anabolic pathways. Recall that biosynthesis requires energy (ATP) and reduction of precursors to which NADPH serves as the source of electrons. So how does the cell determine the use of glucose 6phosphate? Cellular conditions dictate cellular demands. If NADPH is low and must be generated, most animal tissues allow for glucose 6-phosphate to be consumed in the pentose phosphate pathway (also known as the hexose monophosphate shunt or the pentose shunt).This pathway occurs in the cytosol and is divided into two phases. The first phase is the oxidative phase whereby NADPH is generated as glucose 6-phosphate is converted ribulose 5phosphate. The second phase is non-oxidative where three ribulose 5-phosphates are arranged to generate two fructose 6-phosphates and one glyceraldehyde 3-phosphate. Oxidative Phase:

1 Glucose 6-Phosphate + NADP+ + H2O → 1 Ribulose 5-Phosphate + 1 CO2 + 2 NADPH + 2 H+

Non-Oxidative Phase:

3 Ribulose 5-Phosphate → 2 Fructose 6-Phosphate + 1 Glyceraldehyde 3-Phosphate

Net Result:

I.

3-Glucose 6-Phosphate + 6 NADP+ → 2 Fructose 6-Phosphate + Glyceraldehyde 3-Phosphate + 3 CO2 + 6 NADPH + 6 H+

BIG PICTURE – Pentose Phosphate Pathway

There are three irreversible reactions that constitute the oxidative phase where glucose 6-phosphate is converted to ribulose 5-phosphate generating three NADPH and one CO2. The purpose of this half of the pathway is clear, use oxidation of glucose 6-phosphate to generate NADPH for anabolic pathways. The three enzymes used in this phase are glucose 6-phosphate dehydrogenase (produces NADPH), 6phosphogluconolactonase, and 6-phosphogluconate dehydrogenase (produces NADPH).

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Pozzi BICH411 Lecture Supplement Notes The non-oxidative phase is known as the regenerative phase characterized by reversible reactions that catalyze rearrangement of carbon skeletons that produce fructose 6-phosphate and glyceraldehyde 3phosphate that can be fed into other pathways. There are variations of the pentose phosphate pathway that allow for it to be utilized under differing conditions to meet metabolic demands. We will explore these variations after considering mechanistic detail for enzymes involved in the pathway. However, in considering the big picture of glucose metabolism, we see that the pentose phosphate pathway offers an elaborate detour around phosphogluco-isomerase in glycolysis, but serves to generate intermediates that feed back into glycolysis, thus promoting its function while allowing for production of NADPH and ribose 5-phosphate for biosynthesis.

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Pozzi BICH411 Lecture Supplement Notes II.

Mechanisms of Enzymes in the Pentose Phosphate Pathway

The function of this pathway is two-fold, the first phase produces NADPH necessary for anabolic reactions. There are two steps whereby oxidation produces electrons, stored as NADPH (note yellow arrows). The second half of the pathway is non-oxidative and regenerative (see portion in red box below). This part of the pathway utilizes three ribulose 5-phosphate molecules where two- and threecarbon segments are transferred between intermediates. These intermediates play important roles in other carbohydrate catabolic pathways and also in producing ribose 5-phosphate that is necessary for nucleotide synthesis.

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Pozzi BICH411 Lecture Supplement Notes 1. Glucose 6-phosphate dehydrogenase catalyzes the first step of the pentose phosphate pathway whereby glucose 6-phosphate is oxidized to produce 6-phosphoglucono-δ-lactone, transferring electrons from glucose 6-phosphate to NADP+. Glucose 6-phosphate is a cyclic aldehyde structure and with oxidation produces a carboxylic acid. This reaction is rate-limiting and also the first committed step of the pentose phosphate pathway. The enzyme’s function is inhibited by high levels of NADPH, shutting this pathway down when NADPH for synthesis is not needed. 2. 6-Phosphogluconolactonase catalyzes a reaction with 6-phosphoglucono-δ-lactone. The presence of this enzyme serves to accelerate the pathway, as the 6-phosphoglucono-δ-lactone ring structure will spontaneously open and hydrolyze. This is enzyme is necessary as the levels of 6-phosphoglucono-δlactone must be maintained as it is toxic to the cell in too high of concentrations. 3. 6-Phosphogluconate dehydrogenase catalyzes the third reaction in the pentose phosphate pathway that is the final step whereby NADPH is produced, thus it is the final step in the oxidative phase. In this reaction, 6phosphogluconate is oxidatively decarboxylated forming ribulose 5phosphate, so NADPH and CO2 are generated. The enzyme mechanism has two parts, the first step whereby a hydride is transferred to NADP+ from carbon3 of 6phosphogluconate, followed by a decarboxylation event where carbon1 is lost as CO2. A rearrangement produces the product ribulose 5-phosphate. 4. Phosphopentose isomerase catalyzes a reaction (very similar mechanistically to phosphoglucoisomerase) to produce the all-important ribose 5-phosphate. Now in the case of cellular demand where nucleotide, nucleic acid and coenzyme synthesis is necessary this is the product of importance from the pentose phosphate pathway.

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Pozzi BICH411 Lecture Supplement Notes 5. Phosphopentose epimerase catalyzes an acid-base chemistry reaction where the location of an acidic proton is changed. This mechanism involves an active site base that removes a proton from carbon3, producing an enediolate intermediate. The oxyanion formed decomposes, allowing for the formation of the epimer xylulose 5-phosphate. This is the first reaction in a series that involves the rearrangement of carbon skeletons whereby five carbon skeletons are used to construct 3-, 4-, 6-, and 7-carbon skeletons that can be fed into other pathways.

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Transketolase catalyzes the conversion of xylulose 5-phosphate and ribulose 5-phosphate to form glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate. From the basic standpoint, a two-carbon unit from xylulose 5-phosphate is transferred to ribulose 5phosphate producing a 3-carbon unit and a 7-carbon unit. This mechanism utilizes the cofactor thiamine pyrophosphate (TPP), utilizing the basic nature of TPP as it has lost its acidic proton and serves as a good nucleophile (recall we saw this cofactor in alcoholic fermentation catalyzed by the enzyme pyruvate decarboxylase) and attacks the carbonyl of the ketose xylulose 5phosphate. The first product glyceraldehyde 3-phosphate is generated, with the 2-carbon unit possessing a carbonion, that is stabilized by covalent attachment to TPP, serves as a nucleophile to attack the aldose ribose 5phosphate, forming the 7-carbon unit sedoheptulose 7-phosphate and regenerating the ylid form of TPP (negatively charged form).

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Pozzi BICH411 Lecture Supplement Notes 7. Transaldolase allows for conversion of the seven carbon molecule of sedoheptulose 7-phosphate to fructose 6-phosphate. This enzyme utilizes an active site lysyl residue that is used to form a Schiff’s base giving rise to the enamine intermediate. Transaldolase is the enzyme responsible for transferring three carbon units.

III.

Roles of the Pentose Phosphate Pathway

The pentose phosphate pathway can function in different ways to meet the needs of the cell. As such, this pathway can have different variations. 1) More ribose-5-P than NADPH is needed: This is a situation that arises when cells are rapidly dividing and nucleic acids are needed. As the non-oxidative portion of the pathway is reversible the first phase is bypassed and the concentration of intermediates is used to drive the production of ribose 5-phosphate. 2) Both ribose-5-P and NADPH are needed: Ribose 5-phosphate is an important precursor for nucleotide, nucleic acid, and coenzyme synthesis; NADPH is important for donating electrons to reductive anabolic pathways. 3) More NADPH than ribose-5-P is needed: This indicates high amounts of fatty acid biosynthesis occurring. 4) NADPH and ATP are needed, but ribose-5-P is not: Opportunity for pentose phosphate pathway to function by providing energy in form of ATP and NADPH to produce pyruvate when pyruvate is being consumed by the citric acid cycle.

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Pozzi BICH411 Lecture Supplement Notes The following diagram shows the four possible modes for pentose phosphate pathway variations dictated by cellular conditions:

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