NUCLEOTIDE METABOLISM

REVIEW

Nucleotide: has a nitrogenous base, sugar and phosphate group

Nucleoside: has only a bas and a sugar

Bases are purines and pyrimides

Purines (2 ring): Adenine and Guanine

Pyrimidines (1 ring): Cytosine, Thymine, Uracil (RNA)

The sugar is a ribose.

A ribose that has no –OH group on the 2’ carbon is a deoxyribose

INTRODUCTION

Purines and pyrimidines are non-essential. They can be synthesized in the body. The raw materials are readily available from food, but still the DNA and RNA materials are degraded into oligonucleotides when digested so that they can be absorbed and the body has to synthesize them from the raw materials from food. Endonucleases –degrade DNA and RNA into

oligonucleotides Phosphodiesterases -degrade oligonucleotides into

nucleosides Nucleoside phosphorylases –degrade nucleosides in

ribose-1-PO4 and free ribose

After synthesis, these nucleotides are degraded: Purines are degraded into uric acid (insoluble in water

and has the tendency to accumulate in the joints like in gout)

Pyrimidines are degraded into γ-aminoisobutyrate,β -alanine, NH3 and CO2 (they are soluble in water and readily excreted and they may be used as precursors in other pathways. That is why the related diseases to pyrimidines are rare

BIOSYNTHESIS OF PURINE NUCLEOTIDE

Major site of purine synthesis is in the liver.

Three processes that contribute to synthesis:

Synthesis from amphibolic intermediates (de novo pathway)

Phosphoribosylation of purines (salvage pathway) Phosphorylation of purine nucleosides (formation of

monophastes to triphosphates)

Remember that the purine is made of 2 rings. There are different atoms that are donated that come from food:

N1 –comes from ASPARTATE C2 –comes from N10-FORMYL-TETRAHYDROFOLATE N3 and N9 –comes from GLUTAMINE (it is a donor of

both purines and pyrimidines) C4, C5 and N7 comes from GLYCINE C6 comes from RESPIRATORY CO2 C8 –comes from N5,N10-methenyl-tetrahydrofolate

It involves the de novo pathway (raw materials comes from food) and the salvage pathway (recycling).

The most important is the formation of PRPP (5-phosphoribosyl-1-pyrophosphate)

PRPP can be found in both purines and pyrimidines and both de novo and salvage pathway

PRPP becomes Inosine-5-monophosphate (IMP) by a series of 10-11 reactions (depending on what book you read; harper 11- reactions because it included the formation of PRPP in the formation of IMP, lipincott -10 reactions because it did not include formation of PRPP)

PRPP is needed in the 1st

reaction (depends on your reference) of purine synthesis and 5

th reaction in the pyrimidine synthesis

Nucleoside-5-phosphate (ribose-5-phosphate) becomes an activated sugar, PRPP via Phosphoribosyltransferases (PRPP synthetase)

PRPP synthesis requires ATP

According to harper, PRPP synthetase is the rate limiting enzyme while according to lippincott it is not.

For the purpose of the lecture: the rate limiting enzyme for nucleotide metabolism is PRPP synthetase, the rate limiting step in purine synthesis is the 2

nd reaction

FORMATION OF IMP Requires the following: • Five moles of ATP • Two moles of glutamine • One mole of glycine • One mole of CO2 • One mole of aspartate • Two moles of formate:

The formyl moieties are carried on tetrahydrofolate (THF): N5,N10-methenyl-THF N10-formyl-THF

Step 1

The 1st

reaction is catalyzed by PRPP synthase.

The product would be PRPP from α-D-ribose-5-phosphate.

It needs ATP and magnesium

Step 2

The 2nd

reaction is the rate limiting step.

It involves the formation of 5-phospho-β-D-ribosylamine from PRPP.

It is catalyzed by PRPP Glutamyl Amidotransferase.

This enzyme is inhibited by only one end product, GMP.

In this step, glutamine donates Nitrogen 3 and 9.

This step is lacking in RBCs and PMNs (polymorphonuclear cells, neutrophils) that is why they need other tissues to donate purines to them.

Step 3

Catalyzed by glycinamide ribotide synthase

Involves the formation of Glycinamide-5-phophate from 5-phospho-β-o-ribosylamine

Glycine is need in the reaction. It donates the most number of atoms which are C4, C5 and N7

Needs ATP and Magnesium Step 4

Catalyzed by the enzyme, Glycinamide ribotide formyltransferase

Involves the formation of Formylglycinamide ribosyl-5-phosphate

Uses N5,N10-methenyl-THF which donates C8 Step 5

The enzyme used is Formylglycinamide synthethase

Involves the formation of Formylglycinamidine ribosyl-5-phosphate

Also uses glutamine

Needs ATP and magnesium Step 6

Closure of the 5 membered ring

Catalyzed by Aminoimidazole ribotide synthetase

Forms Aminoimidazole ribosyl-5-phosphate Step 7

Carboxylation reaction

Uses Aminoimidazole ribotide carboxylase

Forms Aminoimidazole carboxylate ribosyl-5-phosphate

Respiratory CO2 donates C6 Step 8

Uses Succinylaminoimidazolecarboxamide Ribotide Synthetase

Uses aspartate to donate N1

Forms Aminoimidazole succinyl carboxylate ribosyl-5-phosphate

Aspartate, when it has donated its nitrogen, it becomes fumarate (by product)

Step 9

Uses Adenylpsuccinate lyase

Forms Aminoimidazole carboxamide ribosyl-5-phosphate Step 10

Uses Aminoimidazole Carboxamide Ribotide Formyltransferase

N10-formyl-THF donates C2

Forms Formimidoimidazole carboxamide ribosyl-5-phosphate

Step 11

Closure of the 6 membered ring

Uses IMP Cyclohydrolase

Forms Inosine monophosphate (IMP) Summary of enzymes (memorize) 1. Phosphoribosylpyrophosphate glutamyl amidotransferase (Rate Limiting enzyme) 2. glycinamide ribotide synthetase 3. glycinamide ribotide formyltransferase 4. formylglycinamide synthetase 5. aminoimidazole ribotide synthetase 6. aminoimidazole ribotide carboxylase 7. succinylaminoimidazolecarboxamide ribotide synthetase 8. adenylosuccinate lyase 9. aminoimidazole carboxamide ribotide formyltransferase 10. IMP cyclohydrolase FORMATION OF AMP AND GMP Reaction 1 and 2 in IMP formation are the most important in terms of regulation. IMP is the common pathway for the formation of AMP and GDP. The most important reactions in terms of regulation in the formation of AMP are reaction 12 and 14. Adenylosuccinate synthase (reaction 12) is inhibited by AMP IMP Dehydrogenase (reaction 14) is inhibited by GMP There is also positive regulation. The presence of GMP would increase GTP. GTP stimulates Adenylosuccinate syntase to produce AMP. Similarly, AMP forms ATP, ATP has a positive allosteric effect on IMP dehydrogenase to form GMP. This is known as post regulatory catalytic reaction. Formation of AMP IMP with the presence of Adenylosuccinate synthase together with GTP and Mg forms adenylossucinate. Then it becomes AMP using Adenylosuccinate. Formation of GMP IMP with the presence of IMP Dehydrogenase with NAD as its cofactor (there is reduction) yields Xanthosine monophosphate. It becomes GMP with the use Transamidase. Antifolate drugs (reading assignment yata)

• Azaserine -inhibits step 5 • Diazanorleucine -inhibits step 2 • 6-mercaptopurine -inhibits step formation of AMP and

Xanthosine monophosphate • Mycophonoic acid –inhibits formation of GMP

REGULATION NUCLEOTIDE SYNTHESIS

• The rate of PRPP synthesis depends on the availability of ribose 5-phosphate and on the activity of PRPP synthase, an enzyme sensitive to feedback inhibition by AMP, ADP, GMP, and GDP.

• Increase in PRPP causes gout.

• AMP, GMP, ADP, and GDP inhibits formation of PRPP • AMP and GMP feedback-inhibit adenylosuccinate synthase

and IMP dehydrogenase • Cross regulation between the pathways of IMP

metabolism. GMP increases AMP and vice versa. This means that you cannot increase ATP without increasing GTP and vice versa.

• AMP and GMP also inhibit hypoxanthine-guanine phosphoribosyltransferase (enzyme for the salvage pathway)

• GMP feedback inhibits PRPP glutamyl amidotransferase SALVAGE PATHWAY

• Synthesis of nucleotides from the purine bases and purine nucleosides takes place in a series of steps known as the salvage pathways.

• Free purine bases, adenine, guanine, and hypoxanthine, can be reconverted to their corresponding nucleotides by phosphoribosylation.

• Two key transferase enzymes are involved in the salvage of purines:

1. Adenosine phosphoribosyltransferase (APRT) adenine + PRPP AMP + Ppi

2. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) hypoxanthine + PRPP IMP + Ppi guanine + PRPP GMP + PPi

• 2nd

Salvage Mechanism is the formation of AMP and deoxyCMP from kinase (recycling of nucleosides).

1. Pu-R + ATP ------------ PuR-P +ADP -Nucleoside (Pu-R) with the use of ATP becomes a Nucleotide (PuR-P) by donating its PO4 and becomes ADP

• Adenosine/deoxyadenosine AMP and dAMP

• dC and 2’dG dCMP and dGMP The liver is major site of purine synthesis also provides purines

and nucleosides to tissues incapable of purine synthesis:

A. Human brain tissue has a low level of PRPP glutamyl amidotransferase

• depends in part on exogenous purines. B. Erythrocytes and polymorphonuclear leukocytes

cannot synthesize 5-phosphoribosylamine • utilize exogenous purines to form

nucleotides.

Ribonucleotide reductase complex in synthesis of DNA After the formation of AMP and GMP, we now need triphosphates. The ribonucleotide reductase complex attaches the phosphate bonds to form triphosphates then the formation of DNAs 2’ OHPu/Pyr dNDPs -Reduction of 2’-hydroxyl of purine and pyrimidine ribonucleotides, catalyzed by ribonucleotide reductase complex, forms deoxyribonucleotide diphosphates (dNDPs) The following substrates are needed:

• Thioredoxin • Thioredoxin reductase • NADPH

CATABOLISM *I did not understand the explanation that it is why it is misleading I suggest you listen to the recordings, read the book or follow the diagram

Remember that the purines are made of IMP, hypoxanthine xanthine, adenosine and guanosine. They can be degraded into respective nucleosides by means of nucleotidases. Theyre pathway leads to the formation of guanine and hypoxanthine by means of purine nucleotide phosphorylase (PNP). The next step is the formation of xanthine. Hypoxanthine from oxidase forms xanthine. Also, guanine by means of guanine deaminase forms xanthine. Xanthine by means of xanthine oxidase forms uric acid. Animals do not have gout because of the enzyme uricase. This enzyme is lacking in humans.

PURINE NUCLEOTIDE CYCLE

• States the AMP and IM are continuously being synthesized and degraded

• Synthesis of AMP from IMP and the salvage of IMP via AMP catabolism have the net effect of deaminating aspartate to fumarate.

• Fumarate is the only source of anapleuritic substrate for TCA cycle

• Increases in muscle activity create a demand for an increase in the TCA cycle, in order to generate more NADH for the production of ATP.

AMP by means of AMP deaminase becomes IMP. IMP becomes Adenylosuccinate by the action of aspartate and phosphorylation by GTP. Adenylosuccinate becomes AMP and at the same time removes fumarate. CLINICAL SIGNIFICANCE

BIOSYNTHESIS OF PYRIMIDINE NUCLEOTIDE As similarly to purine synthesis, PRPP is needed but the PRPP in pyrimidine is used in the 5

th reaction (formation of orotic

monophosphate from orotic acid). In purine metabolism PPRP is used in the 2

nd reaction.

Also it needs THF but in pyrimidine, but THF donates hydrogen instead of carbon. The synthesis has 2 parts:

• Formation of UMP • Formation of CTP and TMP

The urea cycle and pyrimidine synthesis uses carbamoyl phosphate synthase II. The difference is that urea cycle is in the mitochondria that is why the enzyme is mitochondrial carbamoyl phosphate synthase II while pyrimidine synthesis the enzyme is called cytosolic carbamoyl phosphate synthase II. The functionality of the 1

st five enzymes of pyrimidine synthesis are

multifunctional catalytic enzymes. In the absence of one enzyme, UMP cannot be formed. They are also called the 5 functional enzymes. The 1

st 3 enzymes acts as one while the 4

th and 5

th also

acts as one enzyme. They are bi functional –another difference from purine synthesis. Step 1

• Catalyzed by Carbamoyl phosphate synthase II (rate limiting enzyme and the enzyme being regulated)

• Carbamoyl phosphate synthase II is inhibited by UTP and activated by ATP and PRPP

• Uses CO2, ATP and the amino acid backbone, Glutamine • Forms Carbamoyl phosphate

Step 2

• Catalyzed by Aspartate transcarbamoylase • This enzyme is inhibited by CTP • Uses aspartic acid • Forms carbamoyl aspartic acid

Step 3

• Catalyzed by Dihydroorotase • Forms Dehydroorotic acid • Involves ring closure

Step 4

• Catalyzed by Dihydroorotate dehydrogenase • Forms Orotic acid • Uses NAD

Step 5

• Catalyzed by Orotate phophoribosyl transferase • Uses PRPP • Forms Orotic monophosphate (OMP)

Disorder Defect Nature of Defect

Clinical Manifestations

Gout 3 different enzyme defects can lead to gout: PRPP synthetase HGPRT

a

glucose-6-phosphatase

activity up deficiency deficiency

hyperuricemia

Lesch-Nyhan syndrome

HGPRT lack of enzyme

Uric acid lithiasis Self mutilation

SCID ADAb lack of

enzyme assignment

Immunodeficiency PNPc lack of

enzyme assignment

Renal lithiasis APRTd lack of

enzyme 2,8-dihydroxyadenine, renal lithiasis

Xanthinuria Xanthine oxidase

lack of enzyme

hypouricemia and xanthine renal lithiasis

von Gierke disease

Glucose-6-phosphatase

enzyme deficiency

Lactic acidosis

Step 6

• Catalyzed by Orotidylic acid decarboxylase • Forms Uridine Monophosphate (UMP)

Step 7

• UMP is phosphorylated to UDP with the use of ATP Step 8

• UDP is again phophorylated to UTP with the use of ATP Step 9

• Catalyzed by CTP synthase • Forms Cytidine Triphosphate (CTP) • Uses Glutamine

Step 10

• Back to UDP, UDP is reduced to deoxyuridine diphosphate (dUDP) by NADPH

• Uses Ribonucleotide reductase Step 11

• dUDP is hydrated to dUMP • a Pi is removed

Step 12

• Catalyzed by thymidylate synthase • Forms Thymidine Monophosphate (TMP) • Uses N5,N10 –methylene THF • Many drugs inhibit formation of TMP in cancer treatment

THF CYCLE

Once thymidylate synthase is activated by N5,N10-methylene THF and has donated its hydrogen, it becomes Dihydrofolate (DHF). DHF is reduced by DHF reductase with NADPH forming THF and NADP. THF is methylated by serine hydroxymethyl transferase into N5,N10-methylene THF.

Relevant drugs The drug 5-fluorouracil (5-FU) binds with dUMP so that it cannot be activated by THF Methotrexate –antifolic acid -folate analogue -inhibitor of dihydrofolate reductase -no formation of THF from DHF SALVAGE PATHWAY thymidine + ATP <——> TMP + ADP deoxyuridine + ATP <——> dUMP + ADP deoxycytidine + ATP <——> dCMP + ADP uracil + ribose-1-phosphate <——> uridine + Pi uridine + ATP ——> UMP + ADP thymine + deoxyribose-1-phosphate <——> thymidine + Pi thymidine + ATP ——> dTMP + ADP DEGRADATION OF PYRIMIDINE NUCLEOTIDES Disorders related to pyrimidine metabolism are rare but fatal. This is

because the products are soluble (β-alanine from metabolism of

CMP and UMP and β-aminoisobutyrate from dTMP), excreted as

CO2, NH3,precursor of acetyl CoA and succinyl CoAfor the Kreb’s

Cycle

Disorders of Pyrimidine Metabolism

Disorder Defective Enzyme Comments

Orotic aciduria,

Type I

orotate phosphoribosyl

transferase and OMP

decarboxylase

assignment

Orotic aciduria,

Type II OMP decarboxylase assignment

Orotic aciduria due

to OTC deficiency

(no hematologic

component)

the urea cycle enzyme,

ornithine transcarbamoylase,

is deficient

increased mitochondrial carbamoyl

phosphate exits and augments

pyrimidine biosynthesis; hepatic

encephalopathy

β-aminoisobutyric

aciduria

transaminase, affects urea

cycle function during

deamination of α-amino acids

to α-keto acids

benign, frequent in Orientals

drug induced orotic

aciduria OMP decarboxylase

allopurinol and 6-azauridine

treatments cause orotic acidurias

without a hematologic component;

their catabolic by-products inhibit

OMP decarboxylase

Regulation of Pyrimidine Biosynthesis

• Aspartate transcarbamoylase, ATCase. o Inhibited by CTP o activated by ATP

• CPS-II domain o activated by ATP o inhibited by UDP, UTP, dUTP, and CTP

• The role of glycine in ATCase regulation is to act as a competitive inhibitor of the glutamine binding site

• ATP levels also regulate pyrimidine biosynthesis at the level of PRPP formation.

• An increase in the level of PRPP results in an activation of pyrimidine synthesis

• OMP decarboxylase: competitively inhibited by UMP at lesser degree, by CMP

• CTP synthase is feedback- o inhibited CTP o activated by GTP.

-END-

Use at your own risk Please check for errors Based from the lecture of Dr. Ricky Gutierrez Trans by Joseph Tayag

-GOODLUCK! :)-

That in all things… God may be glorified….