Understanding the Pentose Phosphate Pathway and Glutathione Function

1. One fate of G6P is the pentose pathway.

2. The pentose pathway is a shunt. • The pathway begins with the glycolytic intermediate glucose 6-P. • It reconnects with glycolysis because two of the end products of the pentose pathway are glyceraldehyde 3-P and fructose 6-P; two intermediates further down in the glycolytic pathway. • It is for this reason that the pentose pathway is often referred to as a shunt.

3. Glycolysis only Moderate glucose flux

4. Pentose Phosphate Pathway Glycolysis Large glucose flux

5. It’s a shunt

6. What does the pentose phosphate pathway achieve? • The pathway yields reducing potential in the form of NADPH to be used in anabolic reactions requiring electrons. • The pathway yields ribose 5-phosphate. • Nucleotide biosynthesis leading to: • DNA • RNA

9. NADPH is a phosphorylated form of NADH. In general, with some exceptions, NADH is used to drive the phosphorylation of ADP to ATP. NADPH is used where reducing potential is required for synthetic reactions.

10. The pentose pathway can be divided into two phases. Non-oxidative interconversion of sugars

11. NADPH + H+ is formed from two separate reactions. The glucose 6-phosphate DH (G6PD) reaction is the rate limiting step and is essentially irreversible. Cells have a greater need for NADPH than ribose 5-phosphate.

12. Regulatory enzyme 5 carbon atoms

14. Regulatory enzyme The enzyme is highly specific for NADP+; the Km for NAD+ is 1000 greater than for NADP+.

18. The nonoxidative phase of the pentose pathway The extensive carbon atom rearrangement. Transketolase requires the coenzyme thiamine pyrophosphate (TPP), the transaldolase does not.

19. Transketolase (TPP) and transaldolase are the link back to glycolysis. • Glyceraldehyde 3-phosphate • Fructose 6-phosphate • Net result: 3C5 2C6 + C3

21. Epimerase inter-converts stereoisomers ribulose-5-P and xylulose-5-P. Isomerase converts the ketose ribulose-5-P to the aldose ribose-5-P. Both reactions involve deprotonation to an endiolate intermediate followed by specific reprotonation to yield the product. Both reactions are reversible.

22. Transketolase & Transaldolase catalyze transfer of 2-C or 3-C molecular fragments respectively, in each case from a ketose donor to an aldose acceptor. D. E. Nicholson has suggested that the names of these enzymes should be changed, since • Transketolase actually transfers an aldol moiety (glycoaldehyde), and • Transaldolase actually transfers a ketol moiety (dihydroxyacetone). However the traditional enzyme names are used here.

23. Transketolase transfers a 2-C fragment from xylulose-5-P to either ribose-5-P or erythrose-4-P. • Transketolaseutilizes as prosthetic group thiamine pyrophosphate (TPP), a derivative of vitamin B1. Pyruvate Dehydrogenase of Krebs Cycle also utilizes TPP as prosthetic group.

24. Transfer of the 2-C fragment to the 5-C aldose ribose-5-phosphate yields sedoheptulose-7-phosphate. • Transfer of the 2-C fragment instead to the 4-C aldose erythrose-4-phosphate yields fructose-6-phosphate.

25. Transaldolase catalyzes transfer of a 3-C dihydroxyacetone moiety, from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate. Transaldolase has an a,b barrel structure.

26. Regulation of the Pentose Pathway • Glucose 6-phosphate DH is the regulatory enzyme. • NADPH is a potent competitive inhibitor of the enzyme. • Usually the ratio NADPH/NADP+ is high so the enzyme is inhibited. • But, with increased demand for NADPH, the ratio decreases and enzyme activity is stimulated.

27. The reactions of the non-oxidative portion of the pentose pathway are readily reversible. • The concentrations of the products and reactants can shift depending on the metabolic needs of a particular cell or tissue.

28. Rapidly dividing cells require more ribose 5- phosphate than NADPH.

29. The need for NADPH and ribose 5-phosphate is balanced.

30. More NADPH is needed than ribose 5-phosphate; Fatty acid synthesis in adipose cells.

31. The cell needs both NADPH and ATP

32. Glutathione and NADPH • What is glutathione? • Why is it important? • How is it related to NADPH?

33. Glutathione is a tripeptide composed of glutamate, cystein, glycine. Reduced glutathione (GSH) maintains the normal reduced state of the cell. Reduced glutathione (GSH)

34. Glutathione Functions -1 • It serves as a reductant. • Conjugates to drugs making them water soluble. • Involved in amino acid transport across cell membranes. • Cofactor in some enzymatic reactions. • rearrangement of protein disulfide bonds.

35. Glutathione Functions -2 • The sulfhydryl of GSH is used to reduce peroxides (ROS) formed during oxygen transport. • Reactive oxygen species (ROS) damage macromolecules (DNA, RNA, and protein) and ultimately lead to cell death. • The resulting oxidized form of GSH is two molecules linked by a disulfide bridge (GSSG).

36. The enzyme glutathione reductase uses NADPH as a cofactor to reduce GSSG back to two moles of GSH. Thus, the pentose pathway is linked to the supply of adequate amounts of GSH.

37. So, what happens if glucose 6-phosphate DH is defective? Insufficient production of NADPH. Which translates into insufficient glutathione. Is this a medical problem? YES

38. Glutathione and Erythrocytes -1 • GSH is extremely important particularly in the highly oxidizing environment of the red blood cell. • Mature RBCs have no mitochondria and are totally dependent on NADPH from the pentose phosphate pathway to regenerate GSH from GSSG via glutathione reductase. • In fact, as much as 10% of glucose consumption, by erythrocytes, is mediated by the pentose pathway.

39. Glutathione and Erythrocytes -2 • The reduced form of glutathione serves as a sulfhydryl buffer. • It maintains cysteine residues in hemoglobin and other proteins in a reduced state. • GSH is essential for normal RBC structure and keeping hemoglobin in Fe++ state.

40. Glutathione and Erythrocytes -3 • Reduced glutathione also detoxifies peroxides. 2GSH + ROOH  GSSG + H2O + ROH • Cells with low levels of GSH are susceptible hemolysis. • Individuals with reduced GSH are subject to hemolysis. • This is often clinically seen as black urine under certain conditions.