Lecture 30 Pyrimidine Metabolism/Disease Raymond B. Birge, PhD

Pyrimidine metabolism (Overview) 1. Nomenclature/nucleotide structure 2. Synthesis pathways 3. Synthesis of deoxy-ribonucleotides 4. Salvage & degradation pathways 5. Metabolic disease of pyrimidine metabolism (orotic aciduria) Suggested reading: Lippencott’s Chapter 22

Structure of Pyrimidines C= 2 oxy, 4 amino T= 2,4 dioxy 5-methyl U= 2,4 dioxy O= 2,4 dioxy 6 carboxy O O O O Orotic acid Uracil NH 2 O Thymine O O Cytosine CH 3 Uracil Orotic Acid

Nomenclature of Pyrimidines * when the base is purine, then the nucleoside ends in OSINE (AdenOSINE, GuanOSINE, InOSINE) when the base is pyrimidine, then the nucleoside ends in IDINE (UrIDINE, CytIDINE, ThymIDINE) PO 4 is an acid: cytidylic acid/cytidylate; note thymidine only deoxyribose Cytidine Monophosphate Cytosine Cytidine BaseNucleotide Base + ribose + P0 4 ester Nucleoside* Base + ribose

Inhibited by UTP; Activated by ATP & PRPP (occurs in cytosol) 1 st Step is Regulated 5-phosphoribosylpyrophosphate

Multifunctional enzyme synthesis: CAD Contrast to purines: Pyrimidines synthesized as free ring C arbamoyl phosphate synthetase II: by UTP; by ATP & PRPP; A spartate transcarbamylase D ihydroorotase 1 polypeptide, 3 domains, 3 activities From Figure in Lippincott Carbamoyl phosphate synthase II Aspartate transcarbamylase Dihyroorotase

Making a pyrimidine From Figure in Lippincott Desaturating the ring gives the pyrimidine, OROTATE. Carbamoyl phosphate synthase II Aspartate transcarbamylase Dihyroorotase Dihyroorotate dehydrogenase

Precursors

Which of the following contributes nitrogen atoms DIRECTLY to both purine and pyrimidine rings? 1.Aspartate 2.Carbamoyl phosphate 3.Bicarbonate 4.Glutamate 5.Tetrahydrofolate

Making UMP: another multifunctional enzyme From Figure in Lippincott UMP synthase: Orotate phosphoribosyl transferase OMP decarboxylase 1 polypeptide, 2 domains, 2 activities OMP decarboxylase Orotate phosphoribosyl transferase Low UMP activity Orotic aciduria (abnormal growth; megaloblastic anemia; treat with uridine-rich diet)

CTP Synthetase ATP, Gln ADP, Pi, Glu Synthesis of CTP Ribose-Tri-P0 4 UTP (Uracil) Ribose-Tri-P0 4 CTP (Cytosine)

Clinical Significance-pyrimidine metabolism ID: A 2 year old female referred to a pediatric clinic Chief Complaint: My baby doesn’t play, sleeps all the time and is weak. History Present Illness: Baby was treated for anemia by family doctor but did not respond to vitamin B 12, folic acid, iron or vitamin C. She is the third-born child of a healthy white couple; her mother had an uneventful pregnancy and a eutopic delivery. Both brothers are healthy. Physical Exam: Low weight and height for age, marked pallor; flacidity & lethargy; sleepiness Pathology: CBC: megaloblastic anemia; UA: increased orotic acid excretion with formation of orotic acid crystals.

Pyrimidine Biosynthesis-IV CPS II ATCDHO DHOD OA OPRT ODC UMP AMP PRPP Purine biosynthesis + _ UTP _ _ Committed Steps Eukaryote Prokaryote

C0 2 + Glutamine + ATP Carbamoyl Phosphate CTP ATP Rate [Aspartate] Carbamoyl Asparate Inhibited by CTP UMP CTP UTP ATCase is feedback inhibited by the end-products of pyrimidine biosynthesis

Ribonucleotides to Deoxyribonucleotides 1.Ribonucleotide Reductase 2.Thymidylate Synthase: (prevent incorporation into RNA)

Ribonucleotide reductase Thioredoxin reductase Inhibited by dATP; Activated by ATP Ribonucleotides to Deoxyribonucleotides

ADP UDP CDP GDP Ribonucleotide Reductase dADP dGDP dCDP dUDP ATP ADP ATP ADP ATP ADP dUTP ATP ADP dUMP H20H20 PPi dTDP dTTP ATP ADP ATP ADP 5,10 THF DHF dATP dGTP dCTP

dUMPTDP N 5,N 10 -methylene- tetrahydrofolate Dihydrofolate Tetrahydrofolate Thymidylate synthase Dihydrofolate reductase NADPH NADP + reduced oxidized Serine transhydroxymethylase Thymidine biosynthesis

Salvage & degradation of pyrimidines uridine-cytidine kinase: nucleoside to nucleotide (deoxycytidine kinase) (thymidine kinase) Salvage: Degradation: pyrimidine rings cleaved and degraded to soluble structures (contrast to purines)

F Uracil 5-Fluoro-Uracil (5FU) 5FU is a simple derivative of Uracil

Targets of drug therapy NADP + N 5,N 10 -methylene- tetrahydrofolate dUMPdTMP Dihydrofolate Tetrahydrofolate Thymidylate synthase Dihydrofolate reductase NADPH reduced oxidized Methotrexate Aminopterin Fluorodeoxyuradylate (5-FU)

Conversion of Serine to Glycine Folate Tetrahydrofolate (FH 4 ) Dihydrofolate reductase N 5, N 10 -Methylene FH 4 Serine Glycine Serine hydroxymethyl transferase (PLP-dep.) Key intermediate in biosynthesis of purines and formation of thymine Important in biosynthesis of heme, porphyrins, and purines

Using nucleotides for selecting hybrid cells

3’ AZido-2’3’ dideoxyThymine (AZT) AZT inhibits HIV reverse transcriptase (RNA-dependent DNA polymerase) This class of compounds (chemotherapeutics, viral inhibitors, etc.) are called nucleoside analogs.

Bottom Line Recognize names and structures of pyrimidines; NMPs/dNTPs Orotate, Uracil, Cytosine, Thymine; CTP/dCTP, TTP Name the sources of atoms in the pyrimidine ring: carbamoyl phosphate (C,N: from Gln, CO 2 ); Aspartate (C,N) Recognize the regulated reaction: Carbamoyl phosphate synthase II: UTP; ATP, PRPP Contrast the synthesis of purines & pyrimidines Explain the cause of Orotic aciduria; Contrast with hyperuricemia Explain mechanisms of the following treatments: sulfonamides, methotrexate, 5-Fluorouracil