: Nitrogen metabolism Part B Nucleotide metabolism

Selected biomolecules derived from aa’s

Nucleotides - general nomenclature AdenineGuanine CytosineUracilThymine Nucleoside Nucleotide monophosphate Nucleotide diphosphate Nucleotide triphosphate Purines Pyrimidines

Nucleotides are generated by salvage of preexisting bases or de novo by assembly from 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 CO 2 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 dihydroorotate which is oxidized to orotate by NAD + Formation of orotate

PRPP (phosphoribosylpyrophosphate) is formed from ribose 5-phosphate and ATP

Orotate couples to PRPP in a reaction driven by the hydrolysis of PP i 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 NH 3 rather than a premade base as in pyrimidine synthesis Catalyzed by glutamine phosphoribosyl amidotransferase

Glycine is coupled to the amino group of phosphoribosylamine N 10 -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 NH 3 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 N 10 -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 + PP i adenine phosphoribosyltransferase Guanine + PRPP  guanylate + PP i Hypoxanthine + PRPP  inosate + PP i 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 synthesis of 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 potent antibacterial 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 balance the two nucleotide pools Carbamoyl phosphate synthetase is also regulated by feedback inhibition

Purine nucleotide biosynthesis is regulated by feedback inhibition at several sites

Ribonucleotide reductase is allosterically regulated 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 NH 3 and CO 2

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

Xanthine oxidase Guanine AdenineXanthine Uric acid Hypoxanthine pKa = 5.75 Crystal deposition & the development of gout Adenine deaminase Guanine deaminase Urate

Lesch-Nyhan syndrome is a consequence of a faulty 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 that drives 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