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

Lecture 30Pyrimidine 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 aminoT= 2,4 dioxy 5-methyl

U= 2,4 dioxyO= 2,4 dioxy 6 carboxy

O

O

O

O

Orotic acid

Uracil

NH2

O

Thymine

O

O

Cytosine

CH3

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)

PO4 is an acid: cytidylic acid/cytidylate; note thymidine only deoxyribose

Cytidine MonophosphateCytosine Cytidine

Base Nucleotide Base + ribose + P04 ester

Nucleoside*Base + ribose

Inhibited by UTP; Activated by ATP & PRPP

(occurs in cytosol)

1st 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 ihydroorotase1 polypeptide, 3 domains, 3 activities

From Figure 22.21 in Lippincott

Carbamoyl phosphate synthase II

Aspartatetranscarbamylase

Dihyroorotase

Making a pyrimidine

From Figure 22.21 in Lippincott

Desaturating the ring gives the pyrimidine,

OROTATE.

Carbamoyl phosphate synthase II

Aspartatetranscarbamylase Dihyroorotase

Dihyroorotatedehydrogenase

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 22.21 in Lippincott

UMP synthase: Orotate phosphoribosyl transferase OMP decarboxylase

1 polypeptide, 2 domains, 2 activities

OMP decarboxylase Orotate phosphoribosyltransferase

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-P04

UTP

(Uracil)

Ribose-Tri-P04

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 didnot respond to vitamin B12, 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 ATC DHO DHOD OA OPRT ODC UMP

AMP

PRPP

PRPP

Purine biosynthesis

+_

UTP_ _

Committed Steps

Eukaryote Prokaryote

C02 + Glutamine + ATP

Carbamoyl Phosphate

CTP

ATP

Rat

e

[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 UDPCDPGDP

Ribonucleotide Reductase

dADP dGDP dCDP dUDP

ATP

ADP

ATP

ADP

ATP

ADP

dUTP

ATP

ADP

dUMP

H20

PPi

dTDP

dTTP

ATP

ADP

ATP

ADP

5,10 THF

DHF

dATP dGTP dCTP

dUMP TDP

N5,N10-methylene-tetrahydrofolate

Dihydrofolate

Tetrahydrofolate

Thymidylate synthase

Dihydrofolate reductase

NADPH

NADP+

reducedoxidized

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+

N5,N10-methylene-tetrahydrofolate

dUMP dTMP

Dihydrofolate

Tetrahydrofolate

Thymidylate synthase

Dihydrofolate reductase

NADPH

reducedoxidized

MethotrexateAminopterin

Fluorodeoxyuradylate (5-FU)

Conversion of Serine to Glycine

N

N

N

NH2N

OH

CH2NHR

H

H

NH

N CH2

NH2C

Folate

Tetrahydrofolate (FH4)

Dihydrofolate reductase

N5, N10-Methylene FH4

NH3+H

CH2OH

CO 2-

C Serine

NH3+H

H

CO 2-

CGlycine

Serine hydroxymethyltransferase (PLP-dep.)

Key intermediatein biosynthesis ofpurines andformation ofthymine 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, CO2 ); 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