1. Final Exam Mon 8 am

2. Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase.
FIGURE Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. Electrons are transmitted (blue arrows) to the enzyme from NADPH via (a) glutaredoxin or (b) thioredoxin. The sulfide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH; GSSG indicates oxidized glutathione). Note that thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group.

3. FIGURE 22-40a Ribonucleotide reductase. (a) Subunit structure
FIGURE 22-40a Ribonucleotide reductase. (a) Subunit structure. Each active site contains two thiols and a group (—XH) that can be converted to an active-site radical; this group is probably the —SH of Cys439, which functions as a thiyl radical.
FIGURE 22-40a Ribonucleotide reductase. (a) Subunit structure. The functions of the two regulatory sites are explained in Figure Each active site contains two thiols and a group (—XH) that can be converted to an active-site radical; this group is probably the —SH of Cys439, which functions as a thiyl radical.

4. binuclear iron center
FIGURE 22-40b Ribonucleotide reductase. (b) The R2 subunits of E. coli ribonucleotide reductase (PDB ID 1PFR). The Tyr residue that acts as the tyrosyl radical is shown in red; the binuclear iron center is orange.
Ribonucleotide reductase. The R2 subunits of E. coli ribonucleotide reductase. The Tyr residue acts as the tyrosyl radical.

5. FIGURE 22-40c Ribonucleotide reductase
FIGURE 22-40c Ribonucleotide reductase. (c) The tyrosyl radical functions to generate the active-site radical (—X･), which is used in the mechanism shown in Figure
The tyrosyl radical functions to generate the active-site radical (—X･)

6. Proposed mechanism for ribonucleotide reductase. In the enzyme of E
Proposed mechanism for ribonucleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the R1 subunit; the active-site radical (—X･) is on the R2 subunit and in E. coli is probably a thiyl radical of Cys439
MECHANISM FIGURE Proposed mechanism for ribonucleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the R1 subunit; the active-site radical (—X･) is on the R2 subunit and in E. coli is probably a thiyl radical of Cys439 (see Figure 22-40).

7. MECHANISM FIGURE (part 6) Proposed mechanism for ribonucleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the R1 subunit; the active-site radical (—X･) is on the R2 subunit and in E. coli is probably a thiyl radical of Cys439 (see Figure 22-40).

8. FIGURE Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site, the substrate-specificity site (right). The diagram indicates inhibition or stimulation of enzyme activity with the four different substrates. The pathway from dUDP to dTTP is described later (see Figure 22-43, 22-44).
Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site, the substrate-specificity site (right). The diagram indicates inhibition or stimulation of enzyme activity with the four different substrates.

9. Biosynthesis of thymidylate (dTMP)
Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase.
FIGURE Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure gives details of the thymidylate synthase reaction.

10. Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase.
Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate.
In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10-methylenetetrahydrofolate (pink and gray).
FIGURE Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10-methylenetetrahydrofolate (pink and gray).

11. Catabolism of purine nucleotides.
FIGURE Catabolism of purine nucleotides. Note that primates excrete much more nitrogen as urea via the urea cycle (Chapter 18) than as uric acid from purine degradation. Similarly, fish excrete much more nitrogen as NH4+ than as urea produced by the pathway shown here.
Catabolism of purine nucleotides.

12. Catabolism of a pyrimidine. Shown here is the pathway for thymine.
The methylmalonylsemialdehyde is further degraded to succinyl-CoA.
FIGURE Catabolism of a pyrimidine. Shown here is the pathway for thymine. The methylmalonylsemialdehyde is further degraded to succinyl-CoA.

13. Allopurinol, an inhibitor of xanthine oxidase
Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine yields the medically effective enzyme inhibitor allopurinol.
At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme.
In addition to blocking uric acid production, inhibition of xanthine oxidase causes an increase in hypoxanthine and xanthine, which are converted to the purines adenosine and guanosine monophosphates. Increased levels of these ribotides causes feedback inhibition of amidophosphoribosyl transferase, the first and rate-limiting enzyme of purine biosynthesis. Allopurinol therefore decreases both uric acid formation and purine synthesis.
FIGURE Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (shaded pink) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme.

15. Azaserine and acivicin, inhibitors of glutamine amidotransferases
Azaserine and acivicin, inhibitors of glutamine amidotransferases. These analogs of glutamine interfere in several amino acid and nucleotide biosynthetic pathways.
FIGURE Azaserine and acivicin, inhibitors of glutamine amidotransferases. These analogs of glutamine interfere in several amino acid and nucleotide biosynthetic pathways.

16. Thymidylate synthesis and folate metabolism as targets of chemotherapy
Thymidylate synthesis and folate metabolism as targets of chemotherapy. During thymidylate synthesis, N5,N10-methylenetetrahydrofolate is converted to 7,8-dihydrofolate; the N5,N10-methylenetetrahydrofolate is regenerated in two steps
FIGURE 22-49a Thymidylate synthesis and folate metabolism as targets of chemotherapy. (a) During thymidylate synthesis, N5,N10-methylenetetrahydrofolate is converted to 7,8-dihydrofolate; the N5,N10-methylenetetrahydrofolate is regenerated in two steps (see Figure 22-44). This cycle is a major target of several chemotherapeutic agents.

17. inhibits thymidylate synthase
FIGURE 22-49b Thymidylate synthesis and folate metabolism as targets of chemotherapy. (b) Fluorouracil and methotrexate are important chemotherapeutic agents. In cells, fluorouracil is converted to FdUMP, which inhibits thymidylate synthase. Methotrexate, a structural analog of tetrahydrofolate, inhibits dihydrofolate reductase; the shaded amino and methyl groups replace a carbonyl oxygen and a proton, respectively, in folate (see Figure 22-44). Another important folate analog, aminopterin, is identical to methotrexate except that it lacks the shaded methyl group. Trimethoprim, a tight-binding inhibitor of bacterial dihydrofolate reductase, was developed as an antibiotic.
inhibits dihydrofolate reductase
aminopterin, is identical to methotrexate except that it lacks the shaded methyl group
Thymidylate synthesis and folate metabolism as targets of chemotherapy

18. MECHANISM FIGURE Conversion of dUMP to dTMP and its inhibition by FdUMP. The left side is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton after step 3. The hydrogens derived from the methylene group of N5,N10-methylenetetrahydrofolate are shaded in gray. The 1,3 hydride shift (step 3), moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. This hydride shift is blocked when FdUMP is the substrate (right). Steps 1 and 2 proceed normally, but result in a stable complexﾑconsisting of FdUMP linked covalently to the enzyme and to tetrahydrofolateﾑthat inactivates the enzyme.

19. MECHANISM FIGURE (part 1) Conversion of dUMP to dTMP and its inhibition by FdUMP. The left side is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton after step 3. The hydrogens derived from the methylene group of N5,N10-methylenetetrahydrofolate are shaded in gray. The 1,3 hydride shift (step 3), moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. This hydride shift is blocked when FdUMP is the substrate (right). Steps 1 and 2 proceed normally, but result in a stable complexﾑconsisting of FdUMP linked covalently to the enzyme and to tetrahydrofolateﾑthat inactivates the enzyme.

20. MECHANISM FIGURE (part 2) Conversion of dUMP to dTMP and its inhibition by FdUMP. The left side is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton after step 3. The hydrogens derived from the methylene group of N5,N10-methylenetetrahydrofolate are shaded in gray. The 1,3 hydride shift (step 3), moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. This hydride shift is blocked when FdUMP is the substrate (right). Steps 1 and 2 proceed normally, but result in a stable complexﾑconsisting of FdUMP linked covalently to the enzyme and to tetrahydrofolateﾑthat inactivates the enzyme.

21. MECHANISM FIGURE (part 3) Conversion of dUMP to dTMP and its inhibition by FdUMP. The left side is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton after step 3. The hydrogens derived from the methylene group of N5,N10-methylenetetrahydrofolate are shaded in gray. The 1,3 hydride shift (step 3), moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. This hydride shift is blocked when FdUMP is the substrate (right). Steps 1 and 2 proceed normally, but result in a stable complexﾑconsisting of FdUMP linked covalently to the enzyme and to tetrahydrofolateﾑthat inactivates the enzyme.

24. Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in E. coli. Regulation of these pathways differs in other organisms.
FIGURE Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in E. coli. Regulation of these pathways differs in other organisms.

25. Methylation of dUMP into dTMP
Thymidylate synthase is a target for some anticancer drugs

26. Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase.
FIGURE Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10-methylenetetrahydrofolate (pink and gray).

27. Catabolism of Purines: Formation of Uric Acid
Excess purine nucleotides are dephosphorylated into nucleosides and phosphate
Adenosine yields hypoxanthine via deamination and hydrolysis
Guanosine yields xanthine via hydrolysis and deamination
Hypoxanthine and xanthine are oxidized into urate, the anion of uric acid
Spiders and other arachnids lack xanthine oxidase

28. FIGURE 22-45 (part 1) Catabolism of purine nucleotides
FIGURE (part 1) Catabolism of purine nucleotides. Note that primates excrete much more nitrogen as urea via the urea cycle (Chapter 18) than as uric acid from purine degradation. Similarly, fish excrete much more nitrogen as NH4+ than as urea produced by the pathway shown here.

29. Catabolism of Purines: Degradation of Urate to Allantoin
Urate is oxidized into a 5-hydroxy-isourate by urate oxidase
Hydrolysis and the subsequent decarboxylation of 5-hydroxy-isourate yields allantoin
Most mammals excrete nitrogen from purines as allantoin
Urate oxidase is inactive in humans and other great apes;
we excrete urate
Birds, most reptiles, some amphibians, and most insects also excrete urate

30. Catabolism of Purines: Degradation of Allantoin
Most mammals do not degrade allantoin
Amphibians and fishes hydrolyze allantoin into allantoate; bony fishes excrete allantoate
Amphibians and cartilaginous fishes hydrolyze allantoate into glyoxylate and urea; many excrete urea
Some marine invertebrates break urea down into ammonia

31. Chapter 22: Summary In this chapter, we learned that:
Some prokaryotes are able to reduce molecular nitrogen into ammonia; understanding details of the nitrogen fixation is one of the holy grails in biochemistry
The twenty common amino acids are synthesized via difficult-to-remember pathways from -ketoglutarate, 3-phospho-glycerate, oxaloacetate, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and ribose-5-phosphate
Nucleotides can be synthesized either de novo from simple precursors, or reassembled from scavenged nucleobases
Purine degradation pathway in most organisms leads to uric acid but the fate of uric acid is species-specific

32. Nucleotide Metabolism Overview

33. HAT medium: Hypoxanthine, aminopterin, thymidine

34. Making monoclonal antibodies

38. HAT Medium

39. “Alfonse, Biochemistry makes my head hurt!!”\