1. Nucleotide Metabolism Lai-Chu Wu, D. Phil. Department of Molecular and Cellular Biochemistry laichu.wu@osumc.edu 1 Dear LSI P1 Learners: This module is Under Construction – please pardon our dust. We will let you know once this version is updated. Thank you for your patience.

2. Lecture Outline I.Pentose phosphate pathway – provides the sugar component of nucleotides II.Metabolism of purine nucleotides – a purine consists of linked five-membered and six-membered rings. Common purines are adenine and guanine III.Metabolism of pyrimidine nucleotides – a pyrimidine consists of a six-membered ring. Common pyrimidines are uracil, cytosine, and thymine IV.Formation of deoxyribonucleotides and synthesis of the thymine nucleotides 2

3. Part I Pentose Phosphate Pathway 3

4. PP Pathway - Learning Objectives Describe the pentose phosphate pathway (including origin of alternative names, functions, substrates, products, tissue/subcellular locations, rate-limiting step/enzyme, and regulation) Differentiate the oxidative pathway from the non-oxidative pathway, and what are their products. Identify the products that participate in nucleotide synthesis or glycolytic pathway Describe the role of NADPH 4

5. The pentose phosphate pathway – and alternate names. The pentose phosphate pathway is part of central metabolism. It is a way by which cells synthesize pentose, and hence the name, although many intermediates are sugars with different carbon lengths. Two products, glyceraldehyde 3-P (GAP) and fructose 6-P (F-6-P), are also intermediates of the glycolytic pathway. It is for this reason the PP pathway is often referred to as the hexose monophosphate shunt. It is also called the phosphogluconate pathway b/c of the intermediate phosphogluconate.

6. Function of the pentose phosphate pathway Provide a source of ribose-5-phosphate for nucleotide and nucleic acid synthesis Provide a route for the use of pentose to form frutose-6-phosphate and glyceraldehyde-3-phosphate Provide a source of NADPH for reductive synthesis (e.g. fatty acids synthesis and dTMP synthesis) and as a reduction agent for antioxidant reactions 6

7. Glutathione-mediated reduction of hydrogen peroxide 7 Hydrogen peroxide is a ROS that damages macromolecules (DNA, RNA, and protein) and ultimately lead to cell death. NADPH, a product of the PP pathway indirectly provides electrons for the reduction of hydrogen peroxide through glutathione. Glutathione (G-SH) is a tripeptide that contains cysteine. The sulfhydryl reduces peroxides to form water. The resulting oxidized form of GSH is two molecules linked by a disulfide bridge (G-S-S-G). A deficiency of glucose-6-P dehydrogenase (the enzyme catalyzing the first reaction in the PP pathway) in turn reduces NADPH, G-SH, and hence integrity of red blood cells and causes hemolytic anemia

8. Location of PP pathway Within the cells, enzymes of the PP pathway are located in the cytoplasm. The pathway is most active in tissues that require NADPH for fatty acid and steroid biosynthesis, and for detoxification. The detoxification, or maintenance of reduced glutathione is practically important in red blood cells, which carry oxygen from tissues to the lungs. The table shows the tissues with active PP pathways 8

9. The PP pathway can be divided into two phases 1.Oxidative phase: –Substrate: glucose-6P and NADP+ –Products: NADPH, ribulose-5P –Controlling enzyme: G-6-P dehydrogenase (a deficiency of this enzyme caused the red blood cell disorder hemolytic anemia) –Regulation: inhibited by NADPH 2.Non-oxidative phase: –Substrate: GAP and Fructose-6P –Products: ribose-5P –Controlling enzyme: none –Regulation: levels of ribose-5P 9

10. The oxidative phase of PP Pathway The oxidative phase of the PP Pathway consists of three reactions: 1.Glucose-6-P is oxidized to 6-phosphogluconolactone to generate one molecule of NADPH 2.Lactone is hydrolyzed to 6-phosphogluconate 3.6-phosphogluconate is oxidatively decarboxylated to ribulose- 5-P with the generation of the second molecule of NADPH. 10 1 2 3

11. Formation of phosphogluconolactone from G6P Glucose-6-phosphate dehydrogenase catalyzes oxidation of the aldehyde (in the form of hemiacetal), at C1 of glucose-6-phosphate to a carboxylic acid in ester linkage (lactone, a cyclic cpd with internal ester bond). NADP + serves as electron acceptor and forms NADPH. This reaction is the committed step of the PP pathway and is strongly inhibited by NADPH and fatty acyl-CoA. The purpose of this is to decrease production of NADPH when its concentration is high, or when the synthesis of fatty acids is no longer necessary. 11

12. Formation of phosphogluconate 6-Phosphogluconolactonase (a lactonase) catalyzes hydrolysis of the ester bond resulting in ring opening, and produces 6- phosphogluconate. Although ring opening occurs in the absence of a catalyst, lactonase speeds up the reaction. Because of the formation of 6-phosphogluconate, the PP pathway is also called phosphogluconate pathway. 12

13. Formation of ribulose-5-P 6-phosphogluconate dehydrogenase catalyzes oxidative decarboxylation of 6-phosphogluconate, to yield the 5-C ketose (a sugar with a ketone group) ribulose-5-phosphate. The hydroxyl at C3 of substrate (becomes C2 of the product; both shown in red circles) is oxidized to a ketone. This promotes loss of the carboxyl at C1 as CO 2. NADP+ again serves as the electron acceptor, and becomes NADPH. 13

14. Non-oxidative phase Non-oxidative phase can further be subdivided into two phases: –Isomerization phase in which ribulose-5-P forms ribose-5-P and xylulose-5-P –Rearrangement phase in which ribose-5-P and xylulose-5-P forms sugars with 4 to 7 carbons, and involves two enzymes, transketolase and transaldolase 14

15. Isomerization phase Isomerase converts the ribulose-5-phosphate to the ribose-5- phosphate. Ribose-5-P contributes to the sugar component of a nucleotide. The enzyme in this reaction is not regulated. However, removal of ribose-5-P will drive the reaction towards its formation Epimerase interconverts the stereoisomers ribulose-5-phosphate and xylulose-5-phosphate, rearranging the –OH linked to a chiral carbon. 15 Ribulose-5-PO 4 Isomerase Nucleotide synthesis Ribulose-5-PO 4 Epimerase

16. Rearrangement Phase The arrangement phase involve two main classes of enzymes, transketolase and transaldolase (click to see reactions involving these enzymes) Ribulose-5-P and xylulose-5-P undergo rearrangement forming a variety of sugars with 3-7 carbons which can be fed into many other metabolic processes. For example, fructose-6-P and glyceraldehyde-3-P are intermediates of glycolysis and erythrose-4-P is a precursor for the biosynthesis of the aromatic amino acids (click). 16 Don’t panic, you need not know all the reactions in detail; see the next 2 slides. 5C 7C 4C 3C 6C

17. Transketolase and transaldolase Transketolase and transaldolase cleaves 2 and 3 carbon units, respectively, from one sugar chain and add to another sugar chain Transaldolase is responsible for cleaving the three carbon unit from a sugar with 7C and adding that 3C unit to glyceraldehyde-3-P thus resulting in sugars with 4C and 6C. Transketolase cleaves a 2C unit from xylulose-5-P and adding that two carbon unit to –ribose-5-P to form glyceraldehyde-3-P and a heptose –erythrose-4-P to form glyceraldehyde-3-P and fructose-6-P 17

18. PP Pathway – summary Names: Pentose phosphate pathway, Hexose monophosphate shunt, phosphogluconate pathway Function: Produce NADPH for reductive biosynthesis and ribose-5-P for nucleotide biosynthesis Connections:To glycolysis through G-6P, GAP, F-6-P Substrates: Oxidative pathway: G-6-P and NADP+ Non-oxidative pathway: GAP and F-6-P Products: Oxidative pathway: NADPH and ribulose-5-P Non-oxidative pathway: ribose-5-P Occurrence:Cytosol of every cells Regulations:Inhibit by a product, NADPH, fatty acyl-CoA Activate by substrates, NADP+ and G-6-P Removal of ribose-5-P 18

19. Part II Purine Metabolism 19

20. Learning Objectives Know the difference between de novo and salvage pathways of purine synthesis Describe the formation and the role of PRPP in purine nucleotide synthesis Know the importance of AMP and GMP synthesis from IMP Know the committing and regulatory steps in purine biosynthesis Compare the degradation of purine nucleotides, AMP and GMP, and how the two pathway converged to form uric acid 20

21. De novo pathway of purine synthesis De novo synthesis of purine is a linear pathway starting with ribose-5- P, ends with IMP and consists of 11 steps. The first two steps will be discuss in the following two slides, and remaining steps briefly in the third slide.

22. Synthesis of PRPP The first step of purine synthesis is the formation of 5-phosphoribosyl- pyrophosphate (PRPP) Ribose-5-P is phosphorylated by PRPP synthetase to PRPP using two phosphates from ATP The enzyme is activated by inorganic phosphate (Pi) and inhibited by its end products, purine ribonucleotides (AMP, ADP, GMP, GDP and IMP). Both the salvage and de novo synthesis pathways of purine and pyrimidine biosynthesis require PRPP 22

23. Synthesis of 5’-phosphoribosylamine In the second reaction of purine biosynthesis, the pyrophosphate group of PRPP is replaced by an amino group from glutamine. This is the committed step in purine biosynthesis. The first step is not the committed step as PRPP can be used for pyrimidine synthesis as well. 23

24. From phosphoribosylamine to IMP Subsequent reactions starting from phosphoribosylamine include adding glycine, adding carbon [from formyl-tetrahydrofolate (THF), adding amine (from glutamine), closing the first ring, adding carboxyl (from CO 2 ), adding aspartate, loss of fumarate, adding carbon (from formyl-THF), and closing the second ring to form inosine monophosphate.

25. Purine biosynthesis – recap from R5P to IMP Synthesis of a purine nucleotide begins with ribose-5-P, the purine ring is built on PRPP, and leads to the first fully formed nucleotide, inosine 5'-monophosphate (IMP). The synthesis of IMP requires simple organic molecules: five moles of ATP, two moles of glutamine, one mole of glycine, one mole of CO 2, one mole of aspartate, and two moles of formate. 25

26. 26 IMP represents a branch point for purine biosynthesis, because it can be converted to either AMP or GMP through two distinct reaction pathways. The pathway leading from IMP to AMP involves addition of amine from aspartate and requires energy from GTP. The pathway leading from IMP to GMP involves an oxidation, addition of amine from glutamine, and requires energy from ATP. IMP forms AMP and GMP by distinct pathways

27. Formation of purine nucleoside diphosphates and triphosphates The conversion of AMP and GMP to their respective nucleoside diphosphate and triphosphate forms can occur in two successive kinase catalyzed steps. In the first step, base-specific kinase phosphorylates each specific nucleoside monophosphate to nucleoside diphosphate In the second step, a single nucleoside diphosphate kinase can phosphorylate any of the four nucleoside diphosphates. 27

28. 28 AMP inhibits conversion of IMP into adenylosuccinate; whereas GMP inhibits the conversion of IMP into XMP. When nucleotide at one pathway is high, and then synthesis from IMP will from nucleotide for the other branch. For example, ATP inhibits its formation but allows GTP to form. The pathway leading to AMP requires energy in the form of GTP; whereas that leading to GMP requires energy in the form of ATP. This reciprocal substrate relationship tends to balance the levels of purine ribonucleotides. Feedback regulation of purine nucleotide synthesis at the IMP branch point IMP adenylosuccinate AMP ADP ATP XMP GMP GDP GTP

29. Regulation of Purine synthesis Purine synthesis is regulated at the first two steps of the de novo pathway. Step 1: Feedback inhibition of PRPP synthetase regulates PRPP levels. This enzyme is inhibited by AMP, ADP, GMP, GDP, IMP and is activated by inorganic phosphate. Step 2: The amidotransferase reaction catalyzed by glutamine PRPP aminotransferase is feed-back inhibited by AMP, GMP and IMP. From IMP, the accumulation of excess ATP leads to accelerated synthesis of GMP, and excess GTP leads to accelerated synthesis of AMP. 29 1. PRPP synthetase 2. Glutamine P RPP synthetase

30. 30 Catabolism of Purine Nucleotides Catabolism of the purine nucleotides leads ultimately to the production of uric acid which is insoluble and is excreted in the urine as sodium urate.

31. Catabolism of Purines: Nucleotides to Bases GMP is hydrolyzed to guanosine which undergoes phosphorolysis to guanine. 31 AMP is degraded by two pathways: An amino group is removed from AMP to form IMP, with IMP further hydrolyzed to inosine AMP is hydrolyzed to adenosine then deaminated to inosine. Inosine is then converted into hypoxanthine by removing the ribose.

32. Catabolism of purine bases form uric acid Hypoxanthine is oxidized by the enzyme xanthine oxidase to xanthine. Guanine is deaminated to xanthine. Xanthine is further oxidized with the production of hydrogen peroxide to form uric acid. 32

33. 33 Salvage of purine bases 1 2 3 The three purine bases, adenine, hypoxanthine, and guanine, formed by purine catabolism or derived from the diet can be converted to nucleotides by the salvage pathway. Salvage pathways is particularly important in those tissues that cannot undergo de novo purine synthesis.

34. Purine Salvage Pathway Two enzymes, HG-PRT and A-PRT, are involved in the purine salvage pathway. HG-PRT converts both hypoxanthine and guanine to their respective nucleoside monophosphates. Ribose-5-P is directly added to the base (hypoxanthine, guanine, and adenine) from PRPP to yield nucleoside monophosphates: 34 Adenine Hypoxanthine guanine AMP IMP GMP Hypoxanthine-guanine Phosphoribosyltransferase (HG-PRT) PRPP PPi PRPP PPi PRPP PPi Adenine phosphoribosyltransferase (A-PRT)

35. Part III Pyrimidine Metabolism 35

36. Objective Know the difference between the de novo and salvage pathways of pyrimidine nucleotide synthesis Describe how the incorporation of PRPP in pyrimidine synthesis is different from that in purine synthesis Describe the degradation of pyrimidine nucleotides. 36

37. De novo synthesis of pyrimidine nucleotides - Overview De novo synthesis of pyrimidine starts with simple organic materials, contains 7 reactions, and produces the first pyrimidine base orotate and then UMP, UDP, UTP and CTP. 37

38. 1: Formation of carbamoyl phosphate Starting materials for pyrimidine biosynthesis includes bicarbonate, amine from glutamine, and phosphate from ATP to form carbamoyl phosphate. The reaction is catalyzed by the cytosolic enzyme carbamoyl phosphate synthetase II (CPS II) and is the committed step in the mammalian pathway. 38

39. 2 and 3: Formation of carbamoyl aspartate and dihyroorotate Carbamoyl phosphate condenses with aspartate to form carbamoyl aspartate. Then, dihydroorotate, a closed 6-membered ring structure is formed by the loss of H 2 O. 39

40. 4 to 6: Formation of UMP, UDP, and UTP Dihydroorotate is oxidized to orotic acid, which in turn combined with PRPP to form orotidine mono-phosphate (OMP). OMP is decarboxylated to form uridine monophosphate (UMP) UDP is formed from UMP by phosphorylation, catalyzed by a specific nucleoside monophosphate kinase, UMP kinase UDP is converted to UTP by another phosphorylation step catalyzed by nucleoside diphosphate kinase, an enzyme with broad specificity for nucleoside diphosphates 40 dihydroorotate NAD+ NADH+/H+ Orotic acid PRPP Ppi orotidine mono-phosphate (OMP) OMP CO2 UMPUDPUTP ATP ADP

41. 7. Formation of CTP 41 UTP CTP CTP is formed from UTP An amino group from glutamine replaces the keto group in uridine triphosphate (UTP) to form cytidine triphosphate (CTP). Note that: UMP does not form CMP, nor does UDP form CDP. UMP and UDP have to be converted to UTP first. glutamine glutamate ATP ADP + Pi

42. Catabolism of Pyrimidines The breakdown of pyrimidines occurs primarily in the liver Catabolism of the pyrimidine nucleotides involves ring opening and forms soluble end-products Cytidine, forms uridine and ultimately to β-alanine Thymine forms β- aminoisobutyrate, and NH 3 and CO 2.

43. Salvage Pathway for Pyrimidine nucleotides 43 Mammalian cells do not possess efficient means of salvaging free bases to pyrimidine nucleotides, but nucleosides are actively phosphorylated to nucleotides by specific kinases: uridine + ATP UMP + ADP cytidine + ATP CMP + ADP Deoxycytidine + ATP dCMP + ADP thymidine + ATP TMP + ADP uridine-cytidine kinase Thymidine kinase deoxycytidine kinase

44. Part IV Formation of deoxyribonucleotides and Synthesis of the Thymine Nucleotides 44

45. Objectives Understand the role of ribonucleotide reductase in converting ribonucleotides to deoxyribonucleotides Identify which phosphorylated form of ribonucleotides can be converted to deoxyribonucleotides Describe the formation of thymine 45

46. Deoxyribonucleotide synthesis - Introduction A cell typically have 5-10 times more RNA than DNA because de novo nucleotide biosynthesis first produces ribonucleotides. However, dNTPs make up our genome and are needed in proliferating cells during DNA replication Ribonucleotides are converted to deoxyribonucleotides, but not all forms can do so Only after ribonucleotides have been converted into the diphosphate form are deoxyribonucleotides produced. 46

47. Ribonucleotide reductase The enzyme ribonucleotide reductase catalyzed the conversion of ribonucleoside diphosphate to 2-deoxyribonucleoside diphosphate In this reaction, the alcohol group on carbon 2 of the ribose sugar is reduced to form deoxyribose NADPH is oxidized to NADP+ (Part I of this lecture described the formation of NADPH by the PP pathway) 47

48. De novo Synthesis of Deoxyribonucleotide Deoxyribonucleotides that are formed by ribonucleotide reductase include dADP, dCDP, dGTP and dUDP. The phosphorylation of dNDPs to dNTPs is catalyzed by nucleoside diphosphate kinases, using ATP as the phosphate donor. Formation of dTTP is shown in the next slide. 48 ADP CDP GDP UDP dADP dCDP dGDP dUDP Catalyzed by Ribonucleotide reductase dATP dCTP dGTP dTTP Catalyzed by nucleoside diphosphokinase dUTP dUDP dUMP dTDP ribonucleoside diphosphates deoxyribonucleoside diphosphates dTMP

49. Formation of dTMP 49 Among uridine nucleotides, only dUMP can be converted to dTMP by the action of thymidylate synthase. The methyl group is donated by methylene tetrahydrofolate (THF), converting THF to dihydrofolate (DHF). In order for the thymidylate synthase reaction to continue, THF must be regenerated from DHF. This is accomplished through the action of dihydrofolate reductase (DHFR). The crucial role of DHFR in thymidine nucleotide biosynthesis makes it an ideal target for chemotherapeutic agents.

50. References Principles of Medical Biochemistry, Meisenberg and Simmons, 3 rd edition, chapter 28. Lippincott’s Illustrated Reviews: Biochemistry, Champe, Harvey, and Ferrier, Chapter 13 and Chapter 22 50

51. Thank you Thank you for completing this module. If you have any questions or comments, please contact me - laichu.wu@osumc.edu 51