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NUCLEOTIDENUCLEOTIDE

METABOLISMMETABOLISMBy:By:

Rebecca Asis Villanueva M.D.Rebecca Asis Villanueva M.D.

Associate ProfessorAssociate ProfessorDepartment of Biochemistry & NutritionDepartment of Biochemistry & Nutrition



NUCLEOTIDENUCLEOTIDE

Ribonucleoside and deoxyribonucleosideRibonucleoside and deoxyribonucleosidephosphatephosphate

Essential for all cellsEssential for all cells

Serve as carriers of activatedServe as carriers of activatedintermediates in the synthesis of someintermediates in the synthesis of somecarbohydrates, lipids and proteinscarbohydrates, lipids and proteins

Structural component of:Structural component of:

coenzyme Acoenzyme AFADFAD

NADNAD

NADPNADP



Important regulatory compounds forImportant regulatory compounds formany pathways of intermediarymany pathways of intermediary

metabolism inhibiting or activatingmetabolism inhibiting or activatingkey enzymes.key enzymes.

³energy currency´ in the cell ³energy currency´ in the cell



Nucleotide structureNucleotide structure

Composed of nitrogenous base, aComposed of nitrogenous base, apentose monosaccharide, and onepentose monosaccharide, and one

two or three phosphate groups.two or three phosphate groups. Nitrogen containing bases are:Nitrogen containing bases are:

purinepurine

pyrimidinepyrimidine



Purine StructurePurine Structure

Guanine



Pyrimidine structurePyrimidine structure

cytosine




thymine




Uracil



DNA and RNA contain:purine bases: adenosine and guanine

pyrimidine base: cytosine

DNA contains thymine as second pyrimidinebase

RNA contains Uracil as second pyrimidine base

Thymine and Uracil differ only by one methylgroup present in thymine but absent on Uracil

i. e. some viral DNA and transfer RNA



Presence of unusual base in a nucleotidesequence:

-aid in its recognition by specificenzyme

- protect it from being degraded bynucleases

Base modification:

methylationhydroxymethylationglycosylationacetylation

alterations of the atoms in pyrimidinering



NucleosidesNucleosides

Addition of pentose sugar to a baseAddition of pentose sugar to a baseproduces a nucleoside.produces a nucleoside.

Adenine AdenosineAdenine AdenosineGuanine GuanosineGuanine Guanosine

Cytosine CytidineCytosine Cytidine

Thymine ThymidineThymine ThymidineUracil UridineUracil Uridine



Sugar:ribose ribonucleoside

2-deoxyribose deoxyribonucleoside

Carbon and nitrogen atoms in the rings of thebase and the sugar are numbered separately

Atoms in the rings of the bases are numbered1-6 in pyrimidine and 1-9 in purines

Carbons in the pentose are numbered 1-5



NucleotidesNucleotides

Mono, di or triphosphate esters of Mono, di or triphosphate esters of nucleosidesnucleosides

Phosphate group attached by linkagePhosphate group attached by linkageto the 5¶ to the 5¶--OH of the pentose is calledOH of the pentose is called

nucleoside 5¶ nucleoside 5¶--phosphate orphosphate or

5¶nucleotide5¶nucleotide

Type of pentose denoted prefix in theType of pentose denoted prefix in thenames ³5¶ ribonucleotide and 5¶ names ³5¶ ribonucleotide and 5¶ deoxyribonucleotide´ deoxyribonucleotide´



Phosphate group are responsible for the

negative changes associated with nucleotidesand nucleic acids

If phosphate group is attached to the 5

carbon of the pentose -Nucleosidemonophosphate (NMP)

i.e. AMP and CMP

If second or third phosphate is added to the

nucleoside - nucleoside diphosphate,triphosphate

i.e. ADP and ATP



Biomedical ImportanceBiomedical Importance

Biosynthesis of purines and pyrimidinesBiosynthesis of purines and pyrimidinesare regulated and coordinated byare regulated and coordinated byfeedback mechanismfeedback mechanism

Production in quantities vary in times,Production in quantities vary in times,which maybe appropriated withwhich maybe appropriated withphysiologic demandsphysiologic demands



Genetic diseases associated withGenetic diseases associated with

purine metabolismpurine metabolism

GoutGout

LeschLesch--Nyhan syndromeNyhan syndrome

Adenosine deaminase deficiencyAdenosine deaminase deficiency Purine nucleoside phosphorylasePurine nucleoside phosphorylase

deficiencydeficiency



Diseases associated withDiseases associated with

pyrimidine catabolismpyrimidine catabolism

Few clinically significant disordersFew clinically significant disorders

Orotic acidemiasOrotic acidemias



Purine and pyrimidine are dietarily Purine and pyrimidine are dietarily

nonessentialnonessential Human tissue synthesize purine andHuman tissue synthesize purine and

pyrimidine from amphibolicpyrimidine from amphibolicintermediatesintermediates



Ingested nucleic acids andIngested nucleic acids and

nucleotidesnucleotides Dietarily nonessentialDietarily nonessential

Degraded in the intestinal tract toDegraded in the intestinal tract to

monosaccharide which may bemonosaccharide which may beabsorbed or converted to purine andabsorbed or converted to purine andpyrimidine bases.pyrimidine bases.



Purine basePurine base

Converted to uric acid by oxidationConverted to uric acid by oxidation

May be absorbed and excreted in theMay be absorbed and excreted in the

urineurine



DE NOVO PURINEDE NOVO PURINENUCLEOTIDENUCLEOTIDE

SYNTHESISSYNTHESIS



Atoms of the purine ring areAtoms of the purine ring arecontributed by:contributed by:

--amino acidsamino acids--CO2CO2

--derivatives of tetrahydrofolatederivatives of tetrahydrofolate



Sources of Individual Atoms inSources of Individual Atoms in

Purine RingPurine Ring

NN

NNN 5N 5



Synthesis of 5Synthesis of 5--phosphoribosylphosphoribosyl--11--

pyrophosphatepyrophosphate Ribose phosphateRibose phosphate

pyrophosphokinase (PRPPpyrophosphokinase (PRPPsynthetase) is activated by Pi andsynthetase) is activated by Pi andinhibited by purine nucleoside di andinhibited by purine nucleoside di andtriphosphates.triphosphates.



Synthesis of 5¶Synthesis of 5¶--

phosphoribosylaminephosphoribosylamine

The amide of glutamine replaces theThe amide of glutamine replaces thepyrophosphate group attached topyrophosphate group attached tocarbon 1 of PRPP. The enzymecarbon 1 of PRPP. The enzyme

glutamine: phosphoribosylglutamine: phosphoribosylpyrophosphate amidotransferase ispyrophosphate amidotransferase isinhibited by the purine 5¶ inhibited by the purine 5¶--nucleotidesnucleotidesAMP,GMP and IMP, the end productsAMP,GMP and IMP, the end products

of this pathway. This is theof this pathway. This is thecommitted step in purine nucleotidecommitted step in purine nucleotidebiosynthesisbiosynthesis



Synthesis of inosineSynthesis of inosine

monophosphate, the ³parent´monophosphate, the ³parent´

purine nucleotidepurine nucleotide

-Requires 4 ATP molecules as an

energy source.



Inhibitors of purine synthesisInhibitors of purine synthesis

specific for inhibiting the growth of specific for inhibiting the growth of rapidly dividing microorganisms( ex.rapidly dividing microorganisms( ex.Sulfonamides )Sulfonamides )



Structural analog s of folic acid (ex.Structural analog s of folic acid (ex.Methotrexate) are useMethotrexate) are usepharmacologically to control thepharmacologically to control thespread of cancer by interfering thespread of cancer by interfering thesynthesis of nucleotides, andsynthesis of nucleotides, andtherefore DNA and RNAtherefore DNA and RNA



Conversion of IMP to AMP andConversion of IMP to AMP and

GMPGMP

The conversion of IMP to either AMPThe conversion of IMP to either AMPor GMP utilizes a twoor GMP utilizes a two--step, energystep, energyrequiring pathway. Note that therequiring pathway. Note that thesynthesis of AMP requires GTP as ansynthesis of AMP requires GTP as anenergy source, whereas theenergy source, whereas thesynthesis of GMP requires ATPsynthesis of GMP requires ATP



the first reaction in each pathway isthe first reaction in each pathway isinhibited by the end product of thatinhibited by the end product of thatpathway. This provides a mechanismpathway. This provides a mechanismfor diverting IMP to the synthesis of for diverting IMP to the synthesis of the species of purine present inthe species of purine present inlesser amounts.lesser amounts.



Conversion of nucleosideConversion of nucleoside

monophosphates to nucleoside dimonophosphates to nucleoside di

and triphosphatesand triphosphates

Nucleoside diphosphates areNucleoside diphosphates are

synthesized from the correspondingsynthesized from the correspondingnucleoside monophosphates by basenucleoside monophosphates by base--specific nucleoside monophosphatespecific nucleoside monophosphatekinases.kinases.



ATP is generally the source of theATP is generally the source of thetransferred phosphate because it istransferred phosphate because it ispresent in higher concentrations thanpresent in higher concentrations thanthe other nucleoside triphosphates.the other nucleoside triphosphates.



For example, adenylate kinase:For example, adenylate kinase:

AMP + ATPAMP + ATP 2 ADP2 ADP

For example, guanylate kinase:For example, guanylate kinase:GMP + ATPGMP + ATP GDP + ADPGDP + ADP



Adenylate kinase is particularlyAdenylate kinase is particularlyactive in liver and muscle, where theactive in liver and muscle, where theturnover of energy from ATP is high.turnover of energy from ATP is high.Its function is to maintain anIts function is to maintain anequilibrium among AMP,ADP, andequilibrium among AMP,ADP, andATP:ATP:

2ADP2ADP AMP + ATPAMP + ATP



Nucleoside diphosphates andNucleoside diphosphates andtriphosphates are interconverted bytriphosphates are interconverted bynucleoside diphosphate kinasenucleoside diphosphate kinase-- ananenzyme that, unlike theenzyme that, unlike themonophosphate kinases, had broadmonophosphate kinases, had broadspecificity.specificity.



For example,For example, GDP + ATPGDP + ATP GTP + ADPGTP + ADP

For example,For example, CDP + ATPCDP + ATP CTP + ADPCTP + ADP



SALVAGE P ATHWAY FORSALVAGE P ATHWAY FOR

PURINEPURINE

Purines from the normal turnover of Purines from the normal turnover of cellular nucleic acids or obtainedcellular nucleic acids or obtainedfrom diet that are not degraded canfrom diet that are not degraded can

be reconverted into nucleosidebe reconverted into nucleosidetriphosphatetriphosphate

Energetically much less expensiveEnergetically much less expensivethan complete de novo synthesisthan complete de novo synthesis

Deficiency causesDeficiency causes LeschLesch--NyhanNyhansyndromesyndrome



Enzymes Involved in SalvageEnzymes Involved in Salvage

PathwayPathway

Adenine phosphoribosyl transferaseAdenine phosphoribosyl transferase

HypoxanthineHypoxanthine--guanineguanine

phosphoribosyl transferasephosphoribosyl transferase

Both utilize PRPP as source of riboseBoth utilize PRPP as source of ribose--55--phosphate groupphosphate group

Release of pyrophosphate makes theseRelease of pyrophosphate makes thesereactions irreversiblereactions irreversible



Salvage PathwaySalvage Pathway

Two key transferase enzymes are involved in the salvage of

purines: adenosine phosphoribosyltransferase (APRT), which

catalyzes the following reaction:

adenine + PRPP <-----> AMP + PPi

and hypoxanthine-guanine phosphoribosyltransferase (HGPRT),

which catalyzes the following reactions:

hypoxanthine + PRPP <------> IMP +

PPi

guanine + PRPP <--------> GMP + PPi



LeschLesch--Nyhan SyndromeNyhan Syndrome

Complete deficiency of Complete deficiency of hypoxanthinehypoxanthine--guanineguaninephosphoribosyl transferasephosphoribosyl transferase

Inability to salvage hypoxanthine orInability to salvage hypoxanthine orguanineguanine

Excessive production of uric acidExcessive production of uric acid

Increased levels of PRPPIncreased levels of PRPP

Decreased IMP and GMPDecreased IMP and GMP

Increased de novo

purine synthesis



DEGRADATION OF PURINEDEGRADATION OF PURINE

NUCLEOTIDESNUCLEOTIDES

Degraded by removal or alterationsDegraded by removal or alterationsof portions of the nucleotideof portions of the nucleotide

End product in human:End product in human:U

ric Acid U

ric Acid Other mammals oxidize uric acid to allantoinOther mammals oxidize uric acid to allantoinwhich can further be degraged to urea orwhich can further be degraged to urea orammoniaammonia

F ti f U i A idF ti f U i A id



Formation of Uric AcidFormation of Uric Acid



Degradation of Dietar y NucleicDegradation of Dietar y Nucleic

Acids in Small Intestine Acids in Small IntestineDNA / RNA

Low pH denatures DNA & RNA

Denatured nucleic acids

nucleases

Oligonucleotides

phosphodiesterases

Mononucleotides nucleotidases Nucleosides nucleosidases

Purines / Primidines

mouth

stomach

small intestine

from pancreas

from pancreas



PYRIMIDINE SYNTHESISPYRIMIDINE SYNTHESIS

Pyrimidine ring synthesized beforePyrimidine ring synthesized beforebeing attached to ribosebeing attached to ribose--55--phosphatephosphate

Sources of carbon and nitrogenSources of carbon and nitrogenatoms:atoms:

GlutamineGlutamine

COCO22

Aspartic acidAspartic acid



Pyrimidine SynthesisPyrimidine Synthesis



Synthesis of Carbamoyl PhosphateSynthesis of Carbamoyl Phosphate

Committed stepCommitted step

Catalyzed by: Carbamoyl phosphateCatalyzed by: Carbamoyl phosphate

synthetaseII

synthetaseII

Inhibited by UTPInhibited by UTP

Activated by ATP and PRPPActivated by ATP and PRPP

cytosoliccytosolic UsesUses --amide group of amine asamide group of amine as

source of nitrogensource of nitrogen



Synthesis of Orotic AcidSynthesis of Orotic Acid

Second step: formation of carbamoylSecond step: formation of carbamoylaspartateaspartate

Catalyzed by aspartate transcarbamoylaseCatalyzed by aspartate transcarbamoylase

Pyrimidine ring closed byPyrimidine ring closed bydihydroorotase resulting todihydroorotase resulting todihydroorotate oxidized to oroticdihydroorotate oxidized to orotic

acidacid



Formation of Pyrimidine NucleotideFormation of Pyrimidine Nucleotide

Complete pyrimidine ring convertedComplete pyrimidine ring convertedto orotidine 5¶ to orotidine 5¶--monophosphatemonophosphate

PRPPPRPP ±± ribose 5ribose 5--phosphate donorphosphate donor

Orotate phosphoribosyl transferaseOrotate phosphoribosyl transferaseproduces OMP with release of produces OMP with release of pyrophosphatepyrophosphate

Biologically irreversibleBiologically irreversible

OMP converted to UMPOMP converted to UMP By Orotidylate decarboxylaseBy Orotidylate decarboxylase

Deficiency: Orotic aciduriaDeficiency: Orotic aciduria



Synthesis of Uridine TriphosphateSynthesis of Uridine Triphosphate

and Cytidine Triphosphateand Cytidine Triphosphate

Amination of UTPAmination of UTP By CTP synthaseBy CTP synthase

NitrogenNitrogen

provided byprovided byglutamineglutamine

Synthesis of CTP from UTP



DEGRADATION OF PYRIMIDINEDEGRADATION OF PYRIMIDINE

NUCLEOTIDESNUCLEOTIDES

Pyrimidine rings can be degraded toPyrimidine rings can be degraded tohighly soluble structureshighly soluble structures

Such asSuch as -- alanine & alanine & --aminoisobutyrateaminoisobutyrate

Can be salvaged and converted intoCan be salvaged and converted intonucleotidesnucleotides

By pyrimidine phosphoribosyltransferaseBy pyrimidine phosphoribosyltransferase

Utilizes PRPP as source of riboseUtilizes PRPP as source of ribose--PP



CONVERSION OF RIBONUCLEOTIDES TOCONVERSION OF RIBONUCLEOTIDES TO

DEOXYRIBONUCLEOTIDESDEOXYRIBONUCLEOTIDES

RIBONUCLEOTIDES

Use as building blocks in RNA synthesis.as nucleotide carriers of other compound

The nucleotides required for DNA synthesis are2· ² deoxyribonucleotides, which are produced fromribonucleoside diphosphate



A. RIBONUCLEOTIDE REDUCTASE A. RIBONUCLEOTIDE REDUCTASE

Ribonucleotide reductaseRibonucleotide reductase (ribonucleoside(ribonucleosidediphosphate reductase )diphosphate reductase )

A multi subunit enzyme ( two identical B1A multi subunit enzyme ( two identical B1subunits and two identical B2 subunits) that issubunits and two identical B2 subunits) that isspecific for reduction of nucleoside diphosphatesspecific for reduction of nucleoside diphosphates(dADP,dGDP,dCDP, and dUDP )(dADP,dGDP,dCDP, and dUDP )

Immediate donors of hydrogen atom needed forImmediate donors of hydrogen atom needed forthe reduction of the 2¶ the reduction of the 2¶--hydroxyl group are twohydroxyl group are two

sulfhydryl groups on the enzyme itself sulfhydryl groups on the enzyme itself²²whichwhichduring rreaction forms a disulfide bondduring rreaction forms a disulfide bond



CONVERSION OF RIBONUCOTIDES TOCONVERSION OF RIBONUCOTIDES TO

DEOXYRIBONUCLEOTIDESDEOXYRIBONUCLEOTIDES

O-P-O-P-P-CH2

H

OO O

O O

O

OH OH

H H

H

BASE

RIBONUCLEOSIDEDIPHOSPHATE

DEOXYRIBONUCLEOSIDE

DIPHOSPHATE

O-P-O-P-O-CH2

H

H H

HOH

BASE

H

O

O O

O O

R ibonucleotide diphosphate

Thioredoxin (2 SH)(reduced)

NADP+

Deoxyribonucleotide diphosphate

Thioredoxin ( S ± S)(oxidized)

NADPH + H+

H20

Ribonucleotide reductase

Thioredoxin reductase



1.Regeneration of reduced enzyme :1.Regeneration of reduced enzyme :

for fibonucleotide reductase to continue to produce deoxyRibonucleotides, the disulfide bong created during production of the2¶-deoxy carbon must be reduced.

The source of reducting equivalents is a peptide co enzyme of Ribonucleotide reductase , THIOREDOXIN.

Thioredoxin ± contains 2 Cysteine residues separated by two aminoacids in peptide chain.

The 2 sulfhydryl groups of thioredoxin donate their hydrogen atoms toribonucleotide reductase in the process of forming a disulfide bond.



2. Regeneration of reduced Thioredoxin :2. Regeneration of reduced Thioredoxin :

Thioredoxin must be converted back to its reduced form inOrder to continue its function.

The necessary equivalents are provided by NADPH + H, andthe reaction is catalyzed by Thioredoxin reductase.



B. REGULATION OF DEOXYRIBONUCLEOTIDEB. REGULATION OF DEOXYRIBONUCLEOTIDE

SYNTHESISSYNTHESIS

Ribonucleotide reductase ± responsible for maintaining a balance supplyOf the deoxyribonuclotides required for DNA synthesis.

to achieve this, the regulation of the enzyme is complex. In additionto the single active site, there are two sites on the enzyme involvedIn regulating this activity.

1. Activity Site- the binding of dATP to an Allosteric site (known as the activity site),

inhibits the overall catalytic activity of the enzyme.

2. Substrate specificity site :- the binding of nucleoside triphosphate to an additional allosteric site

(known as the substrate specificity site ) on the enzyme regulatesSubstrate specificity, causing an increase in the conversion of Ribonucleotides to deoxyribonucleotides as they are required for DNAsynthesis.



REGULATION OF RIBONUCLEOTIDE REDUCTASEREGULATION OF RIBONUCLEOTIDE REDUCTASE

B2subunit

B2subunitR ibonucleoside

Diphosphate(NDP)

DeoxyribonucleosideDiphosphate (dNDP)

B1

subunit

B1

subunit

SUBSTRATE SPECIFICITY SITE

ACTIVITY SITES

SH SH SH SH



SYNTHESIS OF TH YMIDINESYNTHESIS OF TH YMIDINE

MONOPHOSPHATE FROM dUMPMONOPHOSPHATE FROM dUMP

dUMP is converted to dTMP by thymidylate synthetase, w/cUtilizes N5, N10 ± methylene tetrahydrofolate as the source of themethyl group.

this is unusual reaction in that tetrahydrofolate (THF) contributesnot only a carbon unit but also TWO hydrogen atoms for thepteridine ring, resulting in the oxidation of THF to dihydrofolate

(DHF)



Inhibitors of Thymidylate synthetase include thymine analogssuch as 5- fluoracil.

DHF can be reduced to THF by dihyrofolate reductse, an enzymeThat is inhibited in the presence of drugs such as methotrexate.

by decreasing the supply of THF, these folate analogs not onlyInhibit purine synthesis, but by preventing methylation of dUMPto dTMP, they also lower the cellular concentration of this essentialComponent of DNA

DNA synthesis is therefore inhibited and cell growth slowed.



Disorders of PurineDisorders of Purine

Catabolism:Catabolism:I.I. GOUT (Metabolic Disorder of Purine GOUT (Metabolic Disorder of Purine

Catabolism)Catabolism)

Various genetic defects in PRPP synthetaseVarious genetic defects in PRPP synthetasepresent clinically as Gout.present clinically as Gout.

Results in overproduction and overexcretion of Results in overproduction and overexcretion of purine catabolites or resistance to feedbackpurine catabolites or resistance to feedbackinhibition.inhibition.

e.g. An elevated Vmax increased affinity fore.g. An elevated Vmax increased affinity forribose 5ribose 5--phosphate.phosphate.

Gouty ArthritisGouty Arthritis




Catabolism:Catabolism:II.II. LeschLesch-- NyhanNyhan SyndromeSyndrome AnAn overproductionoverproduction hyperuricimiahyperuricimia characterizedcharacterized byby

frequentfrequent episodesepisodes of of uricuric acidacid lithiasislithiasis andand aa bizarrebizarresyndromesyndrome of of self self mutilationmutilation

TheThe accompanyingaccompanying riserise inin intracellularintracellular PRPPPRPP resultsresultsinin purinepurine overproductionoverproduction..

MutationsMutations thatthat decreasedecrease oror abolishabolish hypoxanthinehypoxanthine--

guanineguanine phosphoribosyltransferasephosphoribosyltransferase activityactivity includeincludedeletions,deletions, frameshiftframeshift mutations,mutations, basebase substitutions,substitutions,andand aberrantaberrant mRNAmRNA splicingsplicing..




Catabolism:Catabolism:III.III. VonVon Gierke¶sGierke¶s DiseaseDisease

PurinePurine overproductionoverproduction andand hyperuricemiahyperuricemia(glucose(glucose--66--phosphatasephosphatase deficiency)deficiency) occursoccurs

secondarysecondary toto enhancedenhanced generationgeneration of of thethe PRPPPRPPprecursorprecursor riboseribose 55--phosphatephosphate..

IV.IV. HypouricemiaHypouricemia

HypouricaemiaHypouricaemia andand increasedincreased excretionexcretion of of hypoxanthinehypoxanthine andand xanthinexanthine areare associatedassociatedwithwith xanthinexanthine oxidaseoxidase deficiencydeficiency duedue totogeneticgenetic defectdefect oror toto severesevere liverliver damagedamage..

ff




Catabolism:Catabolism:V.V. AdenosineAdenosine DeaminaseDeaminase & & PurinePurine

NucleosideNucleoside PosphorylasePosphorylase DeficiencyDeficiency AA deficiencydeficiency whichwhich isis associatedassociated withwith anan

immunodeficiencyimmunodeficiency diseasedisease inin whichwhich bothboth thymusthymusderivedderived lymphocyteslymphocytes (T(T cells)cells) andand bonebone marrowmarrow--derivedderived lymphocyteslymphocytes (B(B cells)cells) areare spursespurse andanddysfunctionaldysfunctional..

PurinePurine nucleosidenucleoside phosphorylasephosphorylase deficiencydeficiency isis

associatedassociated withwith aa severesevere deficiencydeficiency of of TT cellscells butbutapparentlyapparently normalnormal BB cellcell functionfunction..

ImmuneImmune dysfunctionsdysfunctions appearappear toto resultresult fromfromaccumulationaccumulation of of dGTPdGTP andand dATP,dATP, whichwhich inhibitinhibitribonucleotideribonucleotide reductasereductase andand therebythereby depletedeplete cellscells of of

DNADNA recursorsrecursors..

Overproduction of Pyrimidine CatabolitesOverproduction of Pyrimidine Catabolites



Overproduction of Pyrimidine CatabolitesOverproduction of Pyrimidine Catabolites

is only rarely associated with clinicallyis only rarely associated with clinically

significant abnormalitiessignificant abnormalities Since the end products of pyrimidine catabolismSince the end products of pyrimidine catabolism

are highly waterare highly water--soluble, pyrimidinesoluble, pyrimidineoverproduction results in few clinical signs oroverproduction results in few clinical signs or

symptoms.symptoms.

In hyperuricemia associated with severeIn hyperuricemia associated with severeoverproduction of PRPP, there is overproductionoverproduction of PRPP, there is overproduction

of pyrimidine nucleotides and increased excretionof pyrimidine nucleotides and increased excretionof ßof ß--alanine. Sincealanine. Since N5 N5 ,, N10N10 ±±methylenemethylene--tetrahydrofolate is required for thymidylatetetrahydrofolate is required for thymidylatesynthesis, disorders of folate and vitamine B12synthesis, disorders of folate and vitamine B12metabolism results in deficiencies of TMP.metabolism results in deficiencies of TMP.

Cli i ll Si ifi tCli i ll Si ifi t



Clinically SignificantClinically Significant

AbnormalitiesAbnormalities

I.I. Orotic AciduriasOrotic Acidurias

TheThe orotic aciduriasorotic acidurias that accompaniesthat accompaniesReye¶s syndromeReye¶s syndrome probably is consequence of probably is consequence of the inability of severely damaged mitochondriathe inability of severely damaged mitochondriato utilize carbamoyl phosphate which thento utilize carbamoyl phosphate which thenbecomes available for cytosolic overproductionbecomes available for cytosolic overproductionof orotic acid.of orotic acid.

Type I orotic aciduriaType I orotic aciduria reflects a deficiency of reflects a deficiency of

both orotate phosphoribosyltransferase andboth orotate phosphoribosyltransferase andorotidylate decarboxylase.orotidylate decarboxylase.

The rarerThe rarer Type II orotic aciduriaType II orotic aciduria is due to ais due to adeficiency only of orotidylate decarboxylase.deficiency only of orotidylate decarboxylase.






II.II. Deficiency of a Urea Cycle Enzyme Deficiency of a Urea Cycle Enzyme results in Excretion of Pyrimidine results in Excretion of Pyrimidine PrecursorsPrecursors

Increased excretion of orotic acid, uracil, andIncreased excretion of orotic acid, uracil, anduridine accompanies a deficiency in liveruridine accompanies a deficiency in livermitochondrialmitochondrial ornithine ornithine transcarbamoylasetranscarbamoylase..

Excess carbamoyl phosphate exits to theExcess carbamoyl phosphate exits to thecytosol, where it stimulates pyrimidinecytosol, where it stimulates pyrimidinenucleotide biosynthesis.nucleotide biosynthesis.

The resultingThe resulting mild orotic aciduriamild orotic aciduria is increasedis increasedby high nitrogen containing food.by high nitrogen containing food.






III.III. Drugs May Precipitate Orotic Drugs May Precipitate Orotic AciduriaAciduria

Allopurinol Allopurinol ,, an alternative substrate foran alternative substrate fororotate phosphoribosyltransferase, competesorotate phosphoribosyltransferase, competes

with orotic acid.with orotic acid. The resulting nucleotide product also inhibitsThe resulting nucleotide product also inhibits

orotidylate decarboxylase, resulting in oroticorotidylate decarboxylase, resulting in oroticaciduria and ortidinuria.aciduria and ortidinuria.

66--Azauridine Azauridine, following conversion to 6, following conversion to 6--azauridylate, also competitively inhibitsazauridylate, also competitively inhibits

orotidylate decarboxylaseorotidylate decarboxylase, enhancing, enhancing

exretion of orotic acid and orotidineexretion of orotic acid and orotidine



INHIBITORS OF PURINE METABOLISMINHIBITORS OF PURINE METABOLISM

(CANCER CHEMOTHERAP Y)(CANCER CHEMOTHERAP Y)

III.III. The turnover of nucleic acids in malignant of itsThe turnover of nucleic acids in malignant of itsformation in these cells may offer a therapeutic value.formation in these cells may offer a therapeutic value.Since most if not. All of these ³inhibitor drugs´ are nonSince most if not. All of these ³inhibitor drugs´ are non--specific, then there is a possibility that it can also affectspecific, then there is a possibility that it can also affectnormal tissues which are rapidly undergoing mitoticnormal tissues which are rapidly undergoing mitotic

cycles like the hematopoietic tissues hence should becycles like the hematopoietic tissues hence should beadministered with proper caution.administered with proper caution.

1.1. 66--Mercaptopurine (6MP)Mercaptopurine (6MP)

This is an antitumor drug which is an analougue of adenineThis is an antitumor drug which is an analougue of adeninemetabolized to a ribonucleotide by the APRT salvagemetabolized to a ribonucleotide by the APRT salvagepathway thus inhibiting the conversion of IMP to GMP orpathway thus inhibiting the conversion of IMP to GMP or

AMP. It also inhibits the rate limiting step of the purineAMP. It also inhibits the rate limiting step of the purinede novo pathway. Simultaneous administration withde novo pathway. Simultaneous administration withallopurinol potentiates its effect as 6allopurinol potentiates its effect as 6--MercaptopurineMercaptopurinewill not be degraded and therefore will delay itswill not be degraded and therefore will delay itsinactivation.inactivation.



INHIBITORS OF PURINE METABOLISMINHIBITORS OF PURINE METABOLISM

(CANCER CHEMOTHERAP Y)(CANCER CHEMOTHERAP Y)

2. Adenosine arabinoside (the sugar is replaced by2. Adenosine arabinoside (the sugar is replaced byan arabinose)an arabinose)

This is used as an antiviral and an antitumor drugThis is used as an antiviral and an antitumor drugin man. It inhibits DNA polymerase after itsin man. It inhibits DNA polymerase after itsconversion to the triphosphate form.conversion to the triphosphate form.

3. Azaserine3. Azaserine

An analogue of glutamine, thus inhibits theAn analogue of glutamine, thus inhibits theincorporation of N3 and N9 into the purineincorporation of N3 and N9 into the purine

ring (de novo biosynthesis), inhibits formationring (de novo biosynthesis), inhibits formationof GMP from IMP, CTP from UTPof GMP from IMP, CTP from UTP--all reactionsall reactionsrequiring the entry of glutamine.requiring the entry of glutamine.

INHIBITORS TO PYRIMIDINE NUCLEOTIDEINHIBITORS TO PYRIMIDINE NUCLEOTIDE



INHIBITORS TO P YRIMIDINE NUCLEOTIDEINHIBITORS TO P YRIMIDINE NUCLEOTIDE

METABOLISM (CANCER AND VIRALMETABOLISM (CANCER AND VIRAL

CHEMOTHERAP Y)CHEMOTHERAP Y)

1.1. Aminopterine/Amithopterine/MethotrexateAminopterine/Amithopterine/Methotrexate

--Inhibits dihydrofolate reductase.Inhibits dihydrofolate reductase.

2. 5 Flurouracil (5 FU)2. 5 Flurouracil (5 FU)

-- An analogue of thymine used in the treatment of solidAn analogue of thymine used in the treatment of solidtumors. It is converted to the monophosphatetumors. It is converted to the monophosphatenucleotide form via the salvage pathway. Eventually itnucleotide form via the salvage pathway. Eventually itis converted to the deoxynucleotide form and binds tois converted to the deoxynucleotide form and binds tothymidylate synthetase, thereby inhibiting thethymidylate synthetase, thereby inhibiting theformation of TMP. As a deoxytriphosphate form, it canformation of TMP. As a deoxytriphosphate form, it canbe incorporated into RNA and inhibits the formation of be incorporated into RNA and inhibits the formation of mature RNA (a step important in translation)mature RNA (a step important in translation)

3. 53. 5--IodouracilIodouracil

-- Functions as an analogue of thymidine, which whenFunctions as an analogue of thymidine, which whenincorporated into DNA bonds with C rather than A, thusincorporated into DNA bonds with C rather than A, thuscausing misreading of the strand.causing misreading of the strand.

INHIBITORS TO PYRIMIDINE NUCLEOTIDEINHIBITORS TO PYRIMIDINE NUCLEOTIDE



INHIBITORS TO P YRIMIDINE NUCLEOTIDEINHIBITORS TO P YRIMIDINE NUCLEOTIDE

METABOLISM (CANCER AND VIRALMETABOLISM (CANCER AND VIRAL

CHEMOTHERAP Y)CHEMOTHERAP Y)

4. Cytosine arabinoside (sugar is replaced to an arabinose)4. Cytosine arabinoside (sugar is replaced to an arabinose)

--used in the treatment of leukemias. It ingibits DNAused in the treatment of leukemias. It ingibits DNApolymerase in its triphosphate form. It has a short half polymerase in its triphosphate form. It has a short half life.life.

REFERENCES:REFERENCES:Daubner, SC et al. Biochemistry. 24:7059Daubner, SC et al. Biochemistry. 24:7059--70657065

Lee L. et al. oligomeric structure of the multifunctional proteinLee L. et al. oligomeric structure of the multifunctional proteinCAD that initiates pyrimidine biosynthesis inCAD that initiates pyrimidine biosynthesis in

mammalian cells. Proc. Nat. Acad of Sci. 1985,mammalian cells. Proc. Nat. Acad of Sci. 1985,82:680282:6802--6806.6806.

Murray, Robert K, et al. Harper¶s review of biochemistry , 25Murray, Robert K, et al. Harper¶s review of biochemistry , 25thth

ed. C. 2000. Appleton and lange. Pp386ed. C. 2000. Appleton and lange. Pp386--401401



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