Pentose Phosphate Pathway An overview of the pathway, its regulation and relationship to glycolysis and other pathways. See chapter 15 of Fundamentals of Biochemisty: Life at the Molecular Level, 4 th Ed by Voet, Voet, and Pratt.
Overview Introduction Stage I Stage II Stage III Transketolase Transaldolase Regulation Relation to other pathways
Introduction Most know that ATP is the energy currency of the cell, but the cell also uses NADH and NADPH for reducing power. Its important to note that while structurally similar, NADH and NADPH are NOT interchangeable in metabolic processes. NADH is used in oxidative phosphorylation to produce ATP and NADPH is used in reductive biosynthesis. The ratio of NAD + /NADH is near 1000 whereas the NADP + /NADPH ratio is near 0.01 under intracellular conditions. NADPH is produced by the oxidation of G6P in the pentose phosphate pathway, also known as the hexose monophosphate shunt. Tissues involved in lipid biosynthesis have a relatively high number of pentose phosphate enzymes. These tissues include: liver, mammary gland, adipose tissues, and adrenal cortex. About 30% of the glucose oxidation of the liver occurs via the pentose phosphate pathway.
Stage I The first stage is a series of reactions that oxidize G6P into ribulose-5-phosphate (Ru5P) with loss of CO 2. NADPH is also produced, this compound now has reducing power to be used in reductive biosynthesis. The reactants of this stage are 3 molecules of G6P, 6 molecules of NADP+ and 3 molecules of water. G6P is considered the starting point of this pathway and may come from a variety of sources including the action of hexokinase on glucose o glycogen breakdown. The products of the this stage are 6 molecules of NADPH, 6 H+, 3 molecules of carbon dioxide molecules, and 3 molecules of Ru5P. The enzymes involved are G6P dehydrogenase, 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase.
Stage I The first reaction is the oxidation of G6P into 6-phosphoglucono-δlactone. This reaction also transfers a hydride ion to NADP + and releases a proton. The enzyme that catalyzes this reaction is glucose-6-phosphate dehydrogenase, an oxidoreductase enzyme. This enzyme is strongly inhibited by NADPH. The second step of this stage is the hydrolysis of the lactone (think ester hydrolysis). The non-enzymatic reaction occurs at an appreciable rate but the enzyme 6-phosphogluconolactonase speeds up the reaction. The third step is the oxidative decarboxylation, catalyzed by 6- phosphogluconate dehydrogenase. This enzyme also uses NADP + to oxidize the compound prior to release of carbon dioxide.
Q: Draw the beta-keto acid intermediate of the 6-phosphogluconoate dehydrogenase reaction Q: Write a reaction scheme showing stage I of the pentose phosphate pathway. Include enzymes and cofactors.
Stage II Stage II involves the isomerization of Ribulose-5-phosphate. Ribulose-5-phosphate isomerase converts ribulose-5-phosphate into ribose-5-phosphate (R5P) (necessary nucleotide precursor) Ru5P can also be converted into Xylulose-5-phosphate (Xu5P) by the action of ribulose-5-phosphate epimerase. Both of the above reactions are similar to TIM and are thought to occur via an enediolate intermediate. The relative amounts of R5P and Xu5P that are produced is dependent on the needs of the cell. In a dividing cell, R5P is needed to make DNA and so mostly R5P will be produced. In a cell that just needs NADPH, R5P and Xu5P will be produced in a 1:2 ratio for conversion to glycolytic intermediates through stage III of the pathway.
Q: Draw the Fischer projections of Ru5P, R5P and Xu5P and indicate how they all differ from one another. Q: Unambiguously define the terms epimer and isomer.
Stage III The last stage of this pathway is the shuffling of carbon atoms to produce compounds that can be used for further metabolism if the necessary product was NADPH and not the R5P for nucleotide synthesis. The two major enzymes involved in this stage are transketolase and transaldolase. Each of which moves carbon from one sugar to another. The reactions of this stage are all reversible so certain sugars can be produced depending on the needs of the cell.
Transketolase This enzyme catalyzes the transfer of two carbon units. Its cofactor is TPP and the proposed mechanism is as follows: 1. TPP ylid adds to the Xu5P carbonyl group. 2. C2-C3 bond cleavage produces GAP and a resonance stabilized carbanion of the C 2 unit. 3. The C 2 carbanion adds to the aldehyde carbon of R5P. 4. TPP is eliminated, producing sedulose-7-phosphate and the free, active enzyme-tpp complex.
Transaldolase This enzyme catalyzes the transfer of a 3 carbon unit between two sugars. The reaction proceeds via an aldol cleavage and uses a Schiff base intermediate. The mechanism is as follows: 1. Schiff base formation. 2. Aldol cleavage. 3. Carbanion addition to carbonyl. 4. Schiff Bass hydrolysis. Since a Schiff base is a necessary intermediate, a lysine residue is likely the major amino acid involved in the active site.
Stage III Overview The first major reaction is the transfer of a C 2 unit from Xu5P to R5P to produce GAP and Sedulose-5-phosphate (S7P). Next, transaldolase moves three carbons from S7P to GAP to produce F6P and Erthrose-4-phosphate (E4P). Then transketolase moves a two carbon unit from Xu5P to E4P to produce another GAP and F6P. So the net yield of the stage is one GAP and two F6P molecules. Remember that one GAP was lost in the transaldolase reaction. For memory purposes its may be helpful to think about how aldolase cleaves F6P into two 3 carbon fragments and so transaldolase moves a 3 carbon fragment. Another memory trick is to know that transketolase moves TWO carbons around.
Q: Draw Fischer projection of the reactants and products in each case. 1. Transaldolase acts on GAP and F6P 2. Transketolase acts on F6P and G6P
Regulation The pathways major products are NADPH and either R5P for nucleotide synthesis or F6P and GAP for glycolysis. The third stage of the hexose monophosphate shunt is utilized when the cell has a higher demand for NADPH then R5P and the third stage is less active when both R5P and NADPH are both needed by the cell. The major control point of the entire pathway is the G6P dehydrogenase (G6PD) enzyme. This enzyme is regulated by the NADP + concentration, if NADPH is being used than NADP + will be present and this will stimulate the pathway to produce NADPH. A deficiency in G6PD is the most common clinical deficiency in the pentose phosphate pathway.
Relation to other Pathways Glucose can be turned into G6P by hexokinase. G6P can then either enter glycolysis or the pentose phosphate pathway. Depending on cellular needs, R5P is produced about halfway through the pentose phosphate pathway. If the cell is synthesizing nucleotides than this will be siphoned off for these purposes. If however the cell does not need R5P then its will undergo further reactions to produce glycolytic intermediates to be used for energy.
Q: Does the ATP yield differ if glucose is first passed through the pentose phosphate pathway versus going directly through glycolysis. Q: How is the flux through the pentose phosphate pathway controlled? Q: Which products are obtained from the pentose phosphate pathway that can t be gotten from glycolysis and what enzymes catalyze the important reactions?