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318 701 1

NUCLEOTIDE METABOLISM

.. Room: 7338E-mail: [email protected]

:

1. Lehninger AL, Nelson DL and Cox MM. Principles of Biochemistry.2nd edition. Irving Place, New York: Worth Publishers, 1993.

2. Mathews CK, van Holde KE and Ahern KG. Biochemistry. 3rd edition.

Sanfrancisco, California: Benjamin/Cummings, an imprint of AddisonWesley Longman, 2000.

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NUCLEOTIDES = repeating units of RNA/DNA

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Nitrogenous bases

Purine bases

Pyrimidine bases

PURINES PYRIMIDINES

DNA contains G, A, T, C

RNA contains G, A, U, C

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NUCLEOTIDES

precursors DNA RNA

ATP GTP essential carriers chemical energy

cofactors NAD, FAD,

S-adenosylmethionine coenzyme A activated biosynthetic

intermediates UDP-glucose CDP-diacylglycerol

cAMP cGMP second messenger

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NUCLEOTIDE METABOLISM

Biosynthesis of Nucleotides

De novo pathway: begins with low-molecular-weight

precursors; amino acids, ribose-5-phosphate, CO2, and NH3

Salvage pathway: synthesizes nucleotides fromnucleosides and bases that become available either in the

diet or through enzymatic breakdown of nucleic acid

Degradation of Nucleotides

intracellular: turnover of unstable mRNA species,DNA repair

extracellular: digestion of nucleic acids ingested in

the diet --> major route by which bases and nucleosidesbecome available for salvage pathway

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Relationships between nucleic acid catabolismand resynthesis of nucleotides by salvage pathway

Nucleic acidcatabolism

Salvage pathways

(nonspecific enzyme)

(Phosphomonoesterases)

(Nucleoside phosphorylases)

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De novo pathways for synthesis and utilization of nucleotides

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PRPP = A central metabolite in de novo andsalvage pathways

PRPP is also an intermediate in histidine and tryptophan biosynthesis.

ATP AMP

PRPP synthetase

PPi

Phosphoribosyltransferase

Deoxyribose analog of PRPP is absent from most cells.-----> phosphoribosyltransferases are not involved directly in

deoxyribonucleotide metabolism

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De Novo Biosynthesis ofPurine Nucleotides

low-molecular-weight precursors to the purine ring

purine ring is assembled while already attached to the ribose-

5-phosphate moiety

PRPP = the first intermediate

inosine 5-monophosphate (IMP) or inosinic acid= the first fully formed purine nucleotide= 5-ribonucleotide of the purine base hypoxanthine

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De novo biosynthesis of the purine ring, from PRPP to inosinic acid

GARsynthetase

GARtransformylase

FGAR amidotransferase

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Potent inhibitors of purine nucleotide synthesis

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De novo biosynthesis of the purine ring, from PRPP to inosinic acid(continued..)

FGAM cyclase

AIR carboxylase

D bi h i f h i i

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De novo biosynthesis of the purine ring,from PRPP to inosinic acid (continued..)

SAICARsynthetase

De novo biosynthesis of the purine ring from PRPP to inosinic acid (continued )

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De novo biosynthesis of the purine ring, from PRPP to inosinic acid (continued..)

SAICAR lyase

De novo biosynthesis of the purine ring from PRPP to inosinic acid (continued )

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AICAR

transformylase

Sulfonamides blockthe synthesis of folatecoenzyme.

D bi h i f h i i f PRPP i i i id ( i d )

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IMPsynthase

Synthesis of AMP and GMP from IMP

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Synthesis ofAMP and GMP from IMP

Xanthinebase

Hypoxanthine

base

Adenylosuccinate

lyase

Adenylosuccinate

synthetaseIMP

dehydrogenase

XMP aminase

Guaninebase Adenine

base

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GDP + ATP GTP + ADP

Nucleoside diphosphatekinase

GMP + ATP GDP + ADPGuanylate kinase

AMP + ATP 2ADPAdenylate kinase

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Feedback control mechanismin the biosynthesis of adenineand guanine nucleotides inE.coli

ADP and GDP are alsorequired for thefeedback regulation.

varies in otherorganisms

Utili ti f Ad i N l tid i

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Utilization ofAdenine Nucleotides inCoenzyme Biosynthesis

Rib fl iFlavin Mononucleotide (FMN)

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Riboflavin, also known asvitamin B2, is acomponent of the flavin

coenzymes, FAD andFMN. It is composed of anisoalloxazine ring systemlinked to ribitol. The ability

of the ring system ofriboflavin to exist as asemiquinone allows theflavin coenzymes to acceptelectrons either singly or in

pairs.

Flavin Mononucleotide (FMN)FMN, also known as riboflavin phosphate, is a

flavin containing electron carrier in the cell. It

participates in oxidation/reduction reactions and,like FAD, differs from the nicotinamide coenzymes

(NAD+ and NADP+) in being able to accept

electrons either singly or in pairs. NAD+

and NADP+ can only accept electrons in pairs.FMNH2 is the reduced form ofFMN.

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FAD is an important acceptor of electrons. FAD is the oxidized form of themolecule (lacks electrons). FADH2 is the reduced form (carries electrons). FAD andFADH2 function in many oxidation reactions, such as those catalyzed by succinatedehydrogenase and fatty acyl-CoA dehydrogenase. Electrons carried by FADH2 do notpass through complex I of the mitochondrial electron transport system and thus donot result in synthesis of as many ATPs in oxidative phosphorylation as electrons fromNADH, which do pass through Complex I.

(FAD)

(FADH2)

NADH is a carrier of electrons produced in biological oxidations The molecule

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NADH is a carrier of electrons produced in biological oxidations. The moleculeexists in two forms that vary in whether or not they are carrying electrons. NADH isthe reduced form of the molecule (carries electrons) and NAD+ is the oxidized form ofthe molecule (lacks electrons). NADH is produced from NAD+ in reactions such as

conversion of acetaldehyde to ethanol by alcohol dehydrogenase. NADH is convertedback to NAD+ by donating electrons (such as in the conversion of pyruvate to lactate)or by depositing electrons into the electron transport system.

Coenzyme A (CoA or CoASH)
http://biochem06/biochem/molex/nadh.htm

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Coenzyme A (CoA or CoASH)Coenzyme A (A for acyl) participates in activation of acyl groups in general,

including the acetyl group derived from pyruvate. The coenzyme is derived

metabolically fromATP, the vitamin pantothenic acid, and -mercaptoethylamine. A free thiol on the last moiety is the functionally significantpart of the coenzyme molecule; the rest of the molecule provides enzyme bindingsites. In acylated derivatives, such as acetyl-coenzyme A, the acyl group is linked tothe thiol group to form an energy-rich thioester. The acylated forms of coenzyme Awill be designated here as acyl-CoA, and the unacylated form as CoA-SH.

Pantothenic acid is a vitamin that forms

an essential part of the acyl-carrier moiety,coenzyme A.

Degradation of Purine Nucleotides

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Degradation of Purine Nucleotides

Nucleotidase

Adenosinedeaminase

Purine nucleosidephosphorylase

Nucleotidase

Purine nucleosidephosphorylase

Guaninedeaminase

Xanthineoxidase

Xanthineoxidase

AMP

Adenosine

NH3

GMP

Guanosine

Inosine Guanine

H2O

H2O

H2O

H2O

H2O

IMP

Pi

Pi

Pi Pi

Pi

NH3

NH3

Ribose-1- P

Ribose-1- P

H2

O2

H2O2O2

O2

Uric acid

Hypoxanthine

Xanthine

Catabolism ofUric acid to ammonia and CO2

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Primates: purine catabolism ends with uric acid

----> excreted

Most animals: uric acid is further catabolized toAllantoin, Allantoic acid, Urea, Ammonia, CO2

---------------> excreted in some fishes

---------------> excreted in most fishes, somemollusks, amphibians

---------------> excreted in some marine invertebrates

Excessive accumulation of Uric acid : Gout

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Excessive accumulation of Uric acid : Gout

overproduction of purine nucleotides----> leading to excessive uric acid synthesis

impaired uric acid excretion through the kidneys

Causes:

3/1000 individuals suffer fromHyperuricemia

Consequences:

Uric acid and its urate salt are quite insoluble Prolonged or acute elevation of blood urate

----> precipitatation of urate, as crystals ofsodium urate,in the synovial fluid of joints

Inflammation (painful arthritis)

Severe degeneration of the joints

Purine-rich foods : liver

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Enzymatic abnormalities in 3 types of gout

Purine rich foods : sweetbreads

e

anchovies

wine

PRPPSynthetase

1

Elevatedlevels

PRPPamido-

transferase

2

Loss offeedbackinhibition

Decreasedlevels

HGPRT = hypoxanthine-guanine phosphoribosyltransferaseAPRT = adenine phosphoribosyltransferase

Note:- gout is also a consequence of chemotherapy- many cases of gout is successfully treated byallopurinol, a structure analog of hypoxanthine

that strongly inhibitsxanthine oxidase----------> accumulation ofhypoxanthine and

xanthine (more soluble than uric acid)

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Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is asalvage pathway enzyme for purine metabolism (another is specific foradenine). When a defect in HGPRT reduces its activity to a low level, gout is

the result. When the defect leads to the complete absence of activity ofHGPRT, Lesch-Nyhan syndrome is the result. The gene for HGPRT isfound on the X chromosome, so the disease is sex-linked. Patients havesevere "gouty" arthritis and a dramatic malfunction of the nervous system,

manifested as behavioral disorders, learning disabilities, and hostile oraggressive behavior, often self-directed. Individuals with Lesch-Nyhansyndrome rarely live beyond 20 years.

Lesch-Nyhan Syndrome

d fi i d f i i b li

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Persons with severe combined immune deficiency (SCID) are totally unable tomount an immune response to antigens. Both the B and T lymphocytes are affected.The disease arises from an inherited lack of a degradative enzyme, adenosinedeaminase (ADA). The reaction shown below illustrates the pathways affected.Lack ofADA allows deoxyadenosine triphophosphate (dATP) to accumulate from

the degradation of DNA. High dATP levels inhibit production of the other dNTPsneeded for DNA replication because of their allosteric effects on the enzymeribonucleotide reductase.

White blood cells are the most affected by lack ofADA. White blood cells must

proliferate for an immune response to occur, and proliferations requires amplesynthesis of DNA and its precursors. Thus, DNA replication is inhibited and whiteblood cells are unable to proliferate, a necessary step for antibody production.

Immunodeficiency and Defective Purine Catabolism

De Novo Biosynthesis ofCarbamoylphosphate

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yPyrimidine Nucleotides

Carbamoylphosphatesynthetase

Aspartatetranscarbamoylase Dihydroorotase Dihydroorotate

dehydrogenase

Orotate phospho-ribosyltransferase

Orotidylatedecarboxylase

UMP kinaseNucleoside diphosphatekinase

CTP synthetase

(= also an intermediate forarginine biosynthesis)

Pyrimidine Catabolism

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Pyrimidine Catabolism

Used in biosynthesisof Coenzyme A

Deoxyribonucleotide rNDP reductase

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DeoxyribonucleotideBiosynthesis

Nucleosidediphosphate kinase

rNDP reductase

rNDP reductase2 2

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22

Either thioredoxin or glutaredoxin can reduce

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Either thioredoxin or glutaredoxin can reducethe oxidized form of rNDP reductase

Biosynthesis of

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ThymineDeoxyribo-

nucleotides

Thymidylate Synthase: A target enzyme for chemotherapy

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Inhibitors ofthymidylatesynthase

Inhibits DNAreplication

Nucleotide Analogs

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3'-Azido-2'3'-Dideoxythymidine (AZT)

: inhibits reverse transcriptase of HIV

Arabinosyladenine (araA)and arabinosylcytosine(araC) are readily converted totriphosphates (araATP and

araCTP). AraATP is a selective

inhibitor of the DNA polymerases

of herpesvirus. araC is used inchemotherapy and functions by

the same mechanism of inhibition

on the cellular polymerase.

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