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Nucleotide Metabolism (and then, you know…)

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• 1865 – Gregor Mendel: proposes the existence of paired factors of heredity

• 1869 – Friedrich Miescher: isolates nuclein and discovers that it was acidic and contained a lot of phosphate

• 1928 – Fred Griffith: observes the transformation of non-pathogenic bacteria through exchange of material with pathogenic ones

• 1944 – Oswald T. Avery, Colin McLeod and Maclyn McCarty: isolate the transforming agent -DNA

• 1950 – Erwin Chargaff: the 1:1 ratio of adenine to thymine and guanine to cytosine

• 1953 – James Watson and Francis Crick (with help from Maurice Wilkins and Rosalind Franklin): identify the double helical structure of DNA

• 1977 – Frederick Sanger: DNA sequencing • 1996 – Ian Wilmut: Dolly the Sheep • 2003 – The Human Genome Project: the sequencing of 3.2

billion base pairs along 23 DNA molecules measuring 2 meters

• 2010 – Craig Venter: synthetic DNA

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BIOSYNTHESIS AND DEGRADATION OF NUCLEOTIDES

• Nucleotides are composed of a nitrogenous base, sugar and phosphate (s)

base and sugar only = nucleoside • Nucleotides are precursors of nucleic acids (DNA, RNA),

energy rich compounds (ATP, GTP, UTP, CTP), coenzymes (NAD/P+ ), FAD, CoA, SAM, Vit B 12) and signaling molecules (cAMP, cGMP)

• Small amounts of nucleic acids in the diet• Pancreatic ribonuclease and deoxyribonuclease change

the nucleic acids into their nucleotide monomers• Intestinal phosphatase converts nucleotides into

nucleosides• The greater portion of nucleosides is modified and

excreted or recycled back to nucleotides

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• The bases of nucleotides are either pyrimidine or purine

• Pyrimidines are characterized by a six-membered heterocyclic aromatic ring with two nitrogens

• Purines have an additional five-membered imidazole ring fused with the aromatic ring of pyrimidines

• The common naturally occurring pyrimidines are cytosine, uracil and thymine (5-methyuracil)

• Purines: cytosine and guanine • Other naturally occurring purine derivatives include

xanthine, hypoxanthine and uric acid

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• The sugar found in ribonucleotides is D-ribose and in deoxyribonucleotides is 2-deoxy D-ribose

• The pentose sugars are attached to the nitrogenous

bases through a β-N-glycosidic bond to give nucleosides

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• In pentoses of nucleosides (and nucleotides), the carbon numbers are given a prime designation (,) to distinguish them from the numbered atoms of the bases

• Nucleosides are named by adding –idine to the root name of pyrimidines and –osine to the root name of purines: cytidine, thymidine, uridine, adenosine, guanosine, inosine

• The prefix deoxy- added in the case of deoxyribonucleosides

Deoxythymidine, deoxyuridine, …

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• Nucleosides are converted to nucleotides through the linking of a phosphate with the 5’ hydroxyl group of ribose or deoxyribose Adenosine 5’-monophosphate (AMP), cytidine 5’

monophosphate (CMP),…• More phosphates could be added to give nucleoside

di- and triphosphates ; or a single phosphate group can be esterified with two hydroxyls of the pentose to give a cyclic nucleotide

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• Two types of pathways lead to the synthesis of nucleotides: the de novo pathways and the salvage pathways

• de novo synthesis begins with amino acids, ribose 5-phosphate and CO2

• Salvage pathways recycle free bases and nucleosides obtained from nucleic acid breakdown (from nucleic acid turnover or from the diet)

• During de novo synthesis new bases are synthesized purine nucleotides are constructed a few atoms at a

time on a ribose-based structure the framework for pyrimidine bases is first synthesized

and is then attached to ribose and modified further • On the other hand, free bases can serve as intermediates

in salvage pathways

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Purine nucleotide synthesisDe novo: is initiated using an activated form of ribose

Ribose-5-phosphate from PPP reacts with ATP to produce 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP)

• The pyrophosphate of PRPP is displaced by the amide nitrogen of glutamine in a reaction catalyzed by glutamine PRPP amidotransferase

The committed step of de novo purine synthesis

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• Once 5-phospho-β-D-ribosylamine is formed, the building of the purine ring begins First, the whole of a glycine molecule is added to

5-phospho-β-D-ribosylamine and phosphoribosylglycinamide is formed

After eight steps, the first purine nucleotide inosine monophosphate (IMP) is produced

Additional nitrogen comes from glutamine and aspartate

N10-formyl THF and CO2 provide additional carbons

Four ATP molecules are used in the process

The sources of the atoms in the purine ring

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• IMP is converted to AMP or GMP: the keto group of IMP is replaced by an amino

group (provided by aspartate) in AMP; requires GTP the dehydrogenation of IMP using NAD+ yields

xanthosine monophosphate (XMP); XMP is then converted to GMP through the donation of the amino group of glutamine; requires ATP

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• Nucleoside triphosphates are the most common types of nucleotide in metabolism

• The production of nucleoside diphosphates requires specific (for type of base) nucleoside monophosphate kinases whereas nucleoside triphosphate formation is catalyzed by nucleoside diphosphate kinases that are not specific for sugar or base

ATP+NMP→ADP+NDPNTPD+NDPA →NDPD+NTPA

The regulation of de novo purine synthesis• The committed step is inhibited by excess levels of IMP,

GMP and ATP• An excess of GMP inhibits the formation of XMP whereas an

excess of AMP inhibits the synthesis of adenylosuccinate • ATP is needed for the synthesis of GMP and GTP is required

for the synthesis of AMP the pools of the two nucleotides are balanced

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• PRPP synthesis is allostericaly inhibited by ADP, GDP and other metabolites from other pathways which use PRPP as a precursor

o Because the de novo pathway consumes much energy, cells synthesize a significant portion of their nucleotides by salvage pathways

Purine salvage pathways• Free purine bases or (deoxy) ribonucleosides can be changed back to mono nucleotides•The liver is the major site of purine nucleotide metabolism; it provides purines and purine nucleosides for other tissues

in some tissues, the de novo pathway produces little or no purine nucleotides – these tissues are dependent upon export from the liver

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• In most tissues AMP is changed by 5’-nucleotidase to adenosine

• Adenosine can then be deaminated by adenosine deaminase to inosine and inosine can then be converted to IMP

• In adenosine deaminase deficiency large concentrations of dATP (because adenosine deaminase acts also on deoxyadenosine) inhibit ribonucleotide reductase; consequently DNA synthesis is depressed

Severe combined immunodeficiency (SCID) - T and B lymphocytes are mainly affected

• Adenosine can also be converted to AMP by adenosine kinase

• Muscular work is accompanied by the production of ammonia, the immediate source of which is AMP

• AMP is changed to IMP with the associated release of NH4+; the

enzyme involved is known as AMP aminohydrolase or AMP deaminase

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• IMP is changed back to AMP through the de novo pathway• The net effect is the generation of fumarate which can

serve an anaplerotic role This is known as the purine nucleotide cycleIt operates in the brain as well as in the muscles

• Adenosine is thought to be a local hormone or autocoidDuring strenuous activity, muscles release

adenosine which has a vasodilatory effect leading to increased delivery of oxygen and nutrients

Adenosine slows the heart rate by blocking the flow of electrical current

•Purine nucleoside phosphorylase converts inosine, guanosine and xanthosine to hypoxanthine, guanine and xanthine, respectively •Deficiency of purine nucleotide phosphorylase affects T cells

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•Purine bases can react with PRPP under the influence of phosphoribosyltransferases • Adenosine phosphoribosyl transferase

catalyzes the reaction of adenine with PRPP to give AMP• Hypoxanthine-guanine phosphoribosyl

transferase (HGPRT) catalyses the reaction of guanine and hypoxanthine with PRPP• The deficiency of HGPRT causes the Lesch-Nyhan

syndrome guanine and hypoxanthine will be degraded

instead of being salvagedmental retardation, self-mutilation

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Purine catabolism•Hypoxanthine is oxidized to xanthine by xanthine oxidase•Guanine is deaminated by guanine aminohydrolase to xanthine• Xanthine is converted to uric acid by xanthine oxidase•Xanthine oxidase is a flavoenzyme with an atom of molybdenum and four iron-sulfur centers; it produces H2O2o in some plants xanthine is trimethylated to caffeine

During periods of extended wakefulness, metabolic activities in the brain lead to the accumulation of adenosine Adenosine induces sleepiness; caffeine promotes

wakefulness by blocking the interaction of extracellular adenosine with neuronal receptors

• In primates uric acid is excreted in the urine as it is• In other mammals urate oxidase changes uric acid to

allantoin; in other organisms allantoin to allantoate to urea to NH4

+

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• Urate is present in the serum of humans at levels near precipitation• This may be because of the use of urate as an anti-oxidant• Gout (from gutta, “drop”) is a common disorder resulting from

hyperuricemia: > 7 mg/dL in males, > 6 mg/dL in females • It causes inflammation of the joints– gouty arthritis

Crystals of sodium urate are deposited in the joints The deposition attracts macrophages that engulf the

crystals Lysosomal membranes of the macrophages are ruptured by

the crystals; this results in the release of lysosomal enzymes and leukotriene B4 into the tissues

Histamine is released from mast cells Visible structures known as tophi may form near joints

causing deformities • The deposition of urate crystals within the renal tubules

causes impaired renal function

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• Primary gout results from genetic defects in purine metabolism

A n overactive PRPP synthase leading to an increase in purine synthesis

Deficiency of HGPRT means less salvage reactions• Glucose-6-phosphatase deficiency leads to the

accumulation of glucose-6-phosphate which will be diverted to ribose-5-phosphate and then to PRPP

• Secondary or acquired gout is caused by various factors that can cause an overproduction of urate or its underexcretion by the kidneys

• Gout can be treated with dietary control and drugs• Allopurinol is a suicide inhibitor of xanthine oxidase

Allopurinol is changed to alloxanthine (oxypurinol) which is a competitive inhibitor of xanthine oxidase

Hypoxanthine and xanthine can be excreted in the urine

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• Allopurinol can also react with PRPP producing allopurinol ribonucleotide thereby reducing purine synthesis• Colchicine inhibits the polymerization of tubulin into

microtubulesThe division and movement of and phagocytosis by white

blood cells is inhibited Lekotriene B4 and histamine release is curtailed

• In earlier times gout was thought to be associated with rich diets and excessive consumption of alcoholic drinks • Lead salts used to be added as preservatives in alcoholic

drinks; pots lined with lead-containing materials • Lead inhibits guanine aminohydrolase leading to the

accumulation of guanine in the joints – Saturnine gout • The bone stores Pb+2 because it is divalent just like Ca+2;

through time Pb+2 is transported to other tissues including the kidney• treatment by chelating agents like

ethylenediaminetetraaceticacid (EDTA)

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The de novo biosynthesis of pyrimidine nucleotides• The synthesis of pyrimidines begins with the formation of

carbamoyl phosphate by CPS II – a cytosolic isozymeUses glutamine as nitrogen donor instead of free

ammonia • Carbamoyl phosphate reacts with aspartate to give

carbamoyl aspartate; the enzyme is aspartate transcarbamoylase (ATCase)• The closure of the pyrimidine ring is then catalyzed by

dehydration through the action of dihydroorotase • Dihyrdroorotate is then oxidized to orotate by

dihydroorotate dehydrogenase ( a flavoprotein) the NADH produced can enter the electron transport chain

• CPS II, aspartate transcarbamoylase and dihydroorotate dehydrogenase are part of a single trifunctional protein known as CAD

• At this point, the basic framework of pyrimidine bases has been constructed

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• Orotate and PRPP react under the catalysis of orotate phosphoribosyl transferase to yield orotidine-5-phosphate (OMP)• OMP is then decarboxylated to uridylate (UMP) by

OMP decarboxylase UMP is phosphorylated to UTP

UTP receives an amide group from glutamine to become cytidine triphosphate (CTP)

• Both orotate phosphoribosyl transferase and OMP decarboxylase activities are located on a protein called UMP synthase

The deficiency of UMP synthase leads to a condition as orotic aciduria - orotate in the urine, retarded growth, anemia

Can be treated with uridine which inhibits the production of orotate and provides building blocks for nucleic acid synthesis

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The sources of the atoms in the pyrimidine ring

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The regulation of de novo pyrimidine synthesis• CPS II is activated by PRPP and inhibited by UTP• As cells approach the S-phase of the cell cycle CPS II

becomes more sensitive to activation by PRPP and less sensitive? to inhibition by UTP phosphorylation of CPS II by MAP kinase at a

specific site leads to an easily activated enzyme while phosphorylation at another site would lead to an easily inhibited enzyme • The ATCase reaction is the committed step of pyrimidine

synthesis; its regulation in bacteria has been discussed under allosteric regulation of enzymes

inhibited by CTP and activated by ATPThe synthesis of thymidylate

• DNA contains thymine instead or uracil• The route to thymidine is through deoxyribonucleotides

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• Deoxyribonucelotides are synthesized from the ribonucleotides by reduction of the 2’-carbon of D-ribose

The enzyme involved is ribonucleotide reductase

The substrates are diphosphonucleotides The reducing equivalents are ultimately donated by

NADPH via thioredoxin or glutaredoxin• UDP is changed to dUDP; dUDP is phosphorylated to

dUTP and then pyrophosphate is removed from dUTP to produce dUMP

• Or dCDP can be changed to dCMP which is deaminated to dUMP

• dUMP receives a methyl group from N5,N10- methylene THF to give dTMP which will be phosphorylated to dTTP

the enzyme is thymidylate synthase

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• The thymidylate synthase reaction converts THF to dihydrofolate (DHF)• THF is regenerated through the action of dihydrofolate

reductase with NADPH providing the reducing equivalents• Rapidly dividing cells require ample supplies of

thymidylate for DNA synthesis; nucleotide biosynthesis can be targeted to design important drugs azaserine is an antibiotic that inhibits glutamine

amidotransferases fluorouracil is changed in the body to

fluorodeoxyridylate (FdUMP) which in turn binds to thymidylate synthase inhibiting it

oExamples of mechanism-based inactivation methotrexate (amethopterin) and aminopterin

are competitive inhibitors of dihydrofolate reductaseo Fluorouracil, methotrexate and aminopterin are

important anticancer agents; trimethoprim- is an antibiotic with a higher affinity for bacterial DHFR

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Pyrimidine salvage pathways• Mammalian cells reutilize a small amount of free

pyrimidine bases• Pyrimidine phosphorylase can synthesize uridine

and cytidine (through the reaction of uracil and cytosine with ribose-1-P) but it cannot use thymine A ribonucleoside containing thymine is not

normally synthesized in the body Instead, thymine will react with deoxyribose-1-

P to form thymidine• The nucleosides uridine, cytidine, thymidine and

deoxycytidine are phosphorylated to their respective nucleotides

Thymidine kinase (TK) levels increase significantly during the S phase of the cell cycle

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Pyrimidine catabolism• The pyrimidine ring can be degraded unlike that of

purines• Cytosine is degraded through uracil Cytidine and deoxycytidine are first deaminated to

uridine and deoxyuridine or: dCMP (deoxycytidylate) can be deaminated to dUMP

(deoxyuridylate) and 5’-nucleotidase converts dUMP to uridine

Salv

age

of p

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idin

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base

s

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• Uridine and cytidine are changed to uracil and cytosine by pyrimidine phosphorylase• dTMP changed to thymine by the successive actions of

5’-nucleotidase and nucleoside phosphorylase • In addition to CO2 and NH4

+ (which enters the urea cycle), the degradation of uracil gives β - alanine and thymine gives β- aminoisobutyrate β - alanine and β-aminoisobutyrate can be excreted Or β - alanine can further be processed to malonyl-

CoA and β-aminoisobutyrate to methylmalonyl-CoAIn some cases β-aminoisobutyrate is produced in

excessive amounts – genetic predisposition (Chinese or Japanese ancestry) and leukemia (massive cell destruction)

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