10-24-11: Nitrogen metabolism Part B

Nucleotide metabolism

Selected biomolecules derived from aa’s

O

OP

OP

OP

O–

OO

–

O

O–

OO–

OH

Base

OH = RiboseH = Deoxyribose

Nucleotides - general nomenclature

N

N H

N N H 2

O

N

R

N

N

N

N H 2

N

R

N

N

N H 2

O

R

N

N H

O

CH 3

O

R

N

N H

O

O

R

Adenine Guanine

Cytosine Uracil Thymine

NucleosideNucleotide monophosphateNucleotide diphosphateNucleotide triphosphate

Purines

Pyrimidines

Nucleotides are generated by salvage of preexisting bases or de novo by assemblyfrom simpler compounds

Pyrimidine biosynthesis

In the de novo synthesis of pyrimidines the ring is synthesized first and then attached to PRPP

From glutamine

Bicarbonate is phosphorylated to form carboxyphosphate, an activated form of CO2

Glutamine is hydrolyzed by carbomyl phosphate synthetase to yield ammonia which reacts with carboxyphosphate to form carbamic acid

Carbamic acid is then phosphorylated to form carbamoyl phosphate

Formation of carbamoyl phosphate is catalyzed by carbamoyl phosphate synthetase

Carbamoyl phosphate reacts with the amino acid aspartate to form carbamoylaspartate. ACTase regulates pyrimidine biosynthesis

Carbamoylaspartate then cyclizes to form dihydroorotatewhich is oxidized to orotate by NAD+

Formation of orotate

PRPP (phosphoribosylpyrophosphate) isformed from ribose 5-phosphate and ATP

Orotate couples to PRPP in a reaction driven by the hydrolysis of PPi

Orotidylate is then decarboxylated to form uridine monophosphate (UMP)

Nucleoside monophosphates are converted nucleoside triphosphates by kinases

UTP can be converted into cytidine triphosphate (CTP) by replacement of a carbonyl group by an amino group from glutamine

ATCase is activated by ATP and inhibited by CTP, the final product of pyrimidine synthesis

Purine biosynthesis

The purine ring is assembled on ribose phosphate

The initial committed step in purine synthesis is displacement of pyrophosphate on

PRPP by NH3 rather than a premade base as in pyrimidine synthesis

Catalyzed by glutamine phosphoribosyl amidotransferase

Glycine is coupled to the amino group of phosphoribosylamine

N10-Formyl-THF transfers a formyl group to the amino group of the glycine residue

The inner amide group is phosphorylated and then converted to an amidine by the addition of NH3 from glutamine

An intramolecular coupling reaction forms a five-membered imidazole ring

Bicarbonate adds first to the exocyclic amino group and then to a carbon atom of the imidazole ring

The imidazole carboxylate is phosphorylated and the phosphoryl group is displaced by the amino group of aspartate

Fumarate leaves followed by the addition of a second formyl group from N10-formyl-THF

Cyclization completes the synthesis of inosinate, a purine nucleotide

AMP and GMP are formed from IMP

Bases are recycled by salvage pathways

Adenine + PRPP adenylate + PPi

adenine phosphoribosyltransferase

Guanine + PRPP guanylate + PPi

Hypoxanthine + PRPP inosate + PPi

hypoxanthine-guanine phosphoribosyltransferase(HGPRT)

Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides

Thymidylate is formed by methylation of dUMP

Tetrahydrofolate is regenerated by dihydrofolate reductase

Several anticancer drugs block synthesisof thymidylate

Rapidly dividing cells require thymidylate for DNA synthesis

Thymidylate synthase is inhibited by fluorouracil, which is converted in vivo to fluorodeoxyuridylate, an analog of dUMP which as a substrate of TMP synthase

irreversibly inhibits the enzyme

Inhibition of regeneration of tetrahydrofolate blocks TMP synthesis

DHF analogs aminopterin and methotrexate are potent competitive inhibiters of dihydrofolate reductase

Methotrexate causes weakening of the immune system; nausea and hair loss are toxic side effects. Why?

Trimethoprim, a folate analog, is a potentantibacterial and antiprotozoal

Trimethoprim binds mammalian dihydrofolate reductase 10,000 less tightly than it does the reductases of

susceptible microorganisms

Pyrimidine nucleotide biosynthesis is regulated by feedback inhibition

Coupling of inhibition by a pyrimidine nucleotide with stimulation by a purine nucleotide helps balancethe two nucleotide pools

Carbamoyl phosphate synthetase is also regulated by feedback inhibition

Purine nucleotide biosynthesis is regulated byfeedback inhibition at several sites

Ribonucleotide reductase is allostericallyregulated at two sites

Nucleotide Degradation

In most living organisms, purines and pyrimidines are constantly being degraded and/or recycled

During digestion nucleases (DNases and RNases) hydrolyze nucleic acids to oligonucleotides of <50 bp

Oligonucleotides are further degraded to free bases and ribose or deoxyribose

Purine catabolism

Generally, dietary purines and pyrimidines are not used in significant amounts to

synthesize cellular nucleic acids

Purines are degraded within enterocytes to uric acid in humans and birds

Pyrimidines are degraded within enterocytes to -alanine or -aminoisobutyric acid, as well as NH3 and CO2

Diseases result from defects in purine catabolic pathways

Adenosine deaminase deficiency results in the depression of DNA synthesis caused by rising dATP levels that

inhibit ribonucleotide reductase

Causes immunodeficiency due to its effect on T and B lymphocytes; associated with SCID - Severe Combined ImmunoDeficiency

SCID is treated by bone marrow transplantation; Adenosine deaminase deficiency was the first disease treated by gene therapy

Gout results from high blood levels of uric acid and recurrent attacks of arthritis

Uric acid loses a proton to form urate, which is normally excreted in the urine

High serum levels of urate (hyperuricemia) cause sodium salts of urate to crystallize and accumulate in joints and kidneys causing inflammation and tissue damage

Allopurinol, an analog of hypoxanthine, is used to treat gout

Allopurinol is a substrate of xanthine oxidase, which convert it to the suicide inhibtor alloxanthine

inhibition of xanthine oxidase causes more soluble xanthine and hypoxanthine to be excreted

Xanthineoxidase

Xanthineoxidase

GuanineAdenine Xanthine

Uric acid

Hypoxanthine

pKa = 5.75

Crystal deposition & the development of gout

Adenine deaminase

Guanine deaminase

NH

NH

N

NH

O

O

O–

Urate

N

N

N

NH

N H 2

NH

N NH

N

O

NH

NH

NH

N

O

NH 2

NH

NH

NH

N

O

O

NH

NH

O

NH

NH

O

O

Lesch-Nyhan syndrome is a consequence of afaulty HGPRT gene

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)is essential for salvage of guanylate and inosinate

Lesch-Nyhan patients have elevated serum urate levels,develop kidney stones and gout

Virtual absence of HGPRT causes elevated PRPP levels thatdrives purine synthesis and the overproduction of urate as unused purines are degraded

Neurological signs include self-mutilation, social aggression,mental deficiency and spasticity

The biochemical basis for the disease remains uncertain

During muscle contratction ATP is converted to ADP. ATP concentrations drop. Adenylate kinase converts 2 ADP to ATP and AMP by moving phosphate groups around

Why is the is reaction beneficial? Beneficial because it rapidly creates ATP with

whatever high E phosphate bonds are around