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3 M1 Renal: ucleotide Metabolism Dr. Robert Lyons Assistant rofessor, Biological Chemistry Director, DA Sequencing Core Web: Fall 2008

4 Amino Acid metabolism Amino acids Folate metabolism Glu, Gln, Asp, 3 Methylene TF Met Cycle Urea oxaloacetate u rin e s DA RA y rim id in e s TCA Cycle fumarate Uric Acid (energy) ucleic Acid metabolism

5 ucleic Acid metabolism Click on any blue rectangle to see details. am i n o a c id s, fo la t e R Carbamoyl hosphate urine Salvage IM urine Biosynthesis yrimidine Biosynthesis M yrimidine Salvage u r in e M T dt Ribonucleotide reductase DA RA Ribonucleotide reductase y r im id in e M dt T urine Degradation yrimidine Degradation 4 ( e n e rg y ) U ric A cid

6 Formation of R: hosphoribose pyrophosphate ribose phosphate AT AM phosphoribose - 1 pyrophosphate R Use in urine Biosynthesis: 2 2 phosphoribose - 1 pyrophosphate glutamine glutamate

7 The First urine: Inosine Monophosphate (folates are involved in this synthesis) Conversion to Adenosine: GT GD + i 2 aspartate ribose-5- Inosine monophosphate Conversion to Guanosine: fumarate ribose-5- Adenosine monophosphate AD + AD AT AM + i ribose-5- Inosine monophosphate ribose-5- gln Xanthosine monophosphate glu 2 ribose-5- Guanosine monophosphate

8 ucleoside Monophosphate Kinases AM + AT <--> 2AD (adenylate kinase) GM + AT <---> GD + AD (guanylate kinase) similar enzymes specific for each nucleotide no specificity for ribonucleotide vs. deoxyribonucleotide

9 Ribonucleotide Reductase X Enzyme AD X Enzyme AD+ ydroxyurea inhibits this enzyme: chemotherapeutic use C 2

10 Regulation of Ribonucleotide Reductase AT CD + (-) dcd dct AT UD + (-) dud dtt GD + dgd dgt AD + dad dat (-)

11 ucleoside Diphosphate Kinase 1 D + 2 T <--> 1 T + 2 D d 1 D + 2 T <--> d 1 T + 2 D o specificity for base o specificity for ribo vs deoxy

12 Feed-forward regulation by R R IM AT GT GM AM GT AT

13 Feed-forward regulation by R R + IM AT GT GM AM GT AT

14 Feed-forward regulation by R R IM AT + + GT GM AM GT AT

15 Feed-forward regulation by R R AT IM - GT GM AM GT AT

16 Degradation of the urine ucleosides: 2 2 ribose Adenosine Guanosine ribose Adenosine deaminase (ADA) i ribose purine nucleoside phosphorylase 2 Inosine ribose Guanine i ribose purine nucleoside phosphorylase Guanine deaminase ypoxanthine Xanthine xanthine oxidase xanthine oxidase Uric acid

17 Salvage athways for urine ucleotides ypoxanthine + R ypoxanthineguanine phosphoribosyl transferase Inosine monophosphate + i ART - Adenine phosphoribosyl transferase - performs a similar function with adenine.

18 Adenosine Deaminase Deficiency: 2 Adenosine deaminase (ADA) 2-deoxyribose Deoxyinosine ypoxanthine Deoxyadenosine Guanine Xanthine dam dad Uric acid dat

19 Gout: deposition of urate crystals in joints, tophi in cooler periphery ypoxanthine Xanthine Uric acid xanthine oxidase xanthine oxidase yperuricemia can be caused by: Accelerated degradation of purines: Accelerated synthesis of purines Increased dietary intake of purines Impaired renal clearance of uric acid Allopurinol inhibits xanthine oxidase and reduces blood uric acid levels: Allopurinol

20 An 80-year-old man with a 30-year history of gout, this patient had been treated intermittently to reduce his serum urate levels. The ew England Journal of Medicine

21 Lesch-yhan Syndrome: Defective GRT hyperuricemia spasticity mental retardation self-mutilation behavior ypoxanthine + R ypoxanthineguanine phosphoribosyl transferase Inosine monophosphate + i A defect in ART does T have similar consequences

22 Myoadenylate Deaminase Fills the TCA Cycle in Muscle 2 3 myoadenylate deaminase 2 ribose-5- ribose-5- Adenosine monophosphate Inosine monophosphate Asp, GT Fumarate GD + i To TCA Cycle

23

24 Carbamoyl phosphate synthetase II - a cytoplasmic enzyme - 2AT + C 3 + glutamine C carbamoyl phosphate 2- + glutamate + 2AD + i used for pyrimidine synthesis C 2 carbamoyl phosphate C 3 + C 2 C aspartate C C C C 2 C C 2 - C 2 orotate

25 rotate is linked to R to form Uridine monophosphate: C 2 orotate C 2 + phosphoribose - 1 pyrophosphate orotidine monophosphate C 2 uridine monophosphate

26 ewly-synthesized uridine monophosphate will be phosphorylated to UD and UT, as described for the purine nucleotides. UT can be converted to CT by CT Synthetase: 2 gln glu uridine triphosphate AT AD i cytidine triphosphate

27 Some UD is converted to dud via ribonucleotide reductase. UD ribonucleotide reductase dud AT AD dut 2 i dum The Thymidylate Synthase Reaction: C 3 thymidylate synthase deoxyuridine monophosphate 5 10, methylene tetrahydrofolate dihydrofolate deoxythymidine monophosphate DFR **** AD methylene donor TF AD+

28 Methotrexate Inhibits Dihydrofolate Reductase: C 3 thymidylate synthase deoxyuridine monophosphate 5 10, methylene tetrahydrofolate dihydrofolate deoxythymidine monophosphate methylene donor TF DFR **** AD+ AD X Methotrexate Dihydrofolate builds up, levels of TF become limiting, thymidylate synthase is unable to proceed. Follow it with a dose of Leucovorin, a.k.a. formyl-tf.

29 FdUM Inhibits The Thymidylate Synthase Reaction: F C 3 5-fluorodeoxyuridine monophosphate (F-dUM) 5 10, methylene tetrahydrofolate thymidylate synthase X dihydrofolate deoxythymidine monophosphate DFR **** AD methylene donor TF AD+

30 Complicated athways for yrimidine roduction: dct CT *** UT dut dtt dcd *** CD UD *** dud dtd UM dum *** dtm de-novo synthesis M This figure is primarily a study aid; you do not need to memorize it or reproduce it. The information here merely summarizes material from previous sections.

31 athologies of pyrimidine nucleotide biosynthesis: rotic acidurea due to TC deficiency - please review your Urea Cycle notes. ereditary orotic acidurea - deficiency of the enzyme that convert orotate to M to UM. ot common.

32 yrimidine degradation: Cytidine deaminase converts cytidine to uridine 2 cytidine deaminase Cytidine Uridine A phosphorylase removes the sugar C 3 C 3 + i pyrimidine phosphorylase thymine + (deoxy)thymidine deoxyribose-1-phosphate Degradation of the base proceeds (products are unimportant here)

33 yrimidines can be salvaged as well: Enzyme: yrimidine nucleoside phosphorylases Thymine + deoxyribose-1-phosphate --> thymidine (T thymidine monophosphate!) C 3 C 3 + i pyrimidine phosphorylase thymine + (deoxy)thymidine deoxyribose-1-phosphate Enzyme: Thymidine kinase - adds the monophosphate back Thymidine + AT --> thymidine monophosphate erpes Simplex Virus carries its own tk gene

34 Certain drugs act via the pyrimidine salvage pathway: G SV thymidine kinase G Acyclovir AcycloGM 5-fluorouracil + F deoxyribose-1-phosphate pyrimidine phosphorylase fluorodeoxyuridine F uridine kinase fluorodeoxyuridine monophosphate (FdUM) F

35 5-FU efficacy depends on rate of degradation vs activation 5-fluorouracil + deoxyribose-1-phosphate F pyrimidine phosphorylase fluorodeoxyuridine F uridine kinase fluorodeoxyuridine monophosphate (FdUM) 5-FU --> --> FdUM + methylene-tf + Thymidylate Synthase F --> inactivation of TS Degradation (via dihydropyrimidine dehydrogenase, DD DD inhibitors can potentiate 5FU activity

36 Capecitabine mode of action: C 3 F dealkylation cytidine deaminase pyrimidine phoshorylase F fluorouracil capecitabine Cytosine arabinoside (arac) activation and inactivation: Uridine arabinoside (IACTIVE) cytidine deaminase 2 Cytosine arabinoside (arac) cytidine kinase 2 Cytosinearabinoside monophosphate (ACTIVE)

37 Additional Source Information for more information see: Slide 4: Robert Lyons Slide 5: Robert Lyons Slide 6: Robert Lyons Slide 7: Robert Lyons Slide 9: Robert Lyons Slide 10: Robert Lyons Slide 12: Robert Lyons Slide 13: Robert Lyons Slide 14: Robert Lyons Slide 15: Robert Lyons Slide 16: Robert Lyons Slide 17: Robert Lyons Slide 18: Robert Lyons Slide 19: Robert Lyons Slide 20: The ew England Journal of Medicine, Slide 21: Robert Lyons Slide 22: Robert Lyons Slide 24: Robert Lyons Slide 25: Robert Lyons Slide 26: Robert Lyons Slide 27: Robert Lyons Slide 28: Robert Lyons Slide 30: Robert Lyons Slide 32: Robert Lyons Slide 33: Robert Lyons Slide 34: Robert Lyons Slide 35: Robert Lyons Slide 36: Robert Lyons