Biosynthesis of nucleotides Natalia Tretyakova, Ph.D. Phar 6152 Spring 2004 Required reading: Stryer’s Biochemistry 5 th edition, p , (or Stryer’s Biochemistry 4 th edition p , )

Tentative Lecture plan: Biosynthesis of Nucleotides Introduction. Biological functions and sources of nucleotides. Nucleotide metabolism Biosynthesis of pyrimidine ribonucleotides Biosynthesis of purine ribonucleotides Biosynthesis of deoxyribonucleotides. Inhibitors of nucleotide metabolism as drugs Review 04-12Exam

Biological functions and sources of nucleotides. Nucleotide metabolism Required reading: Stryer’s Biochemistry 5 th Ed., p ,

Biological functions of nucleotides 1. Building blocks of nucleic acids (DNA and RNA). 2. Involved in energy storage, muscle contraction, active transport, maintenance of ion gradients. 3. Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine). 4. Components of coenzymes (NAD +, NADP +, FAD, FMN, and CoA) 5. Metabolic regulators: a. Second messengers (cAMP, cGMP) b. Phosphate donors in signal transduction (ATP) c. Regulation of some enzymes via adenylation and uridylylation

Nucleotides  -glycosidic bond RNA- ribose (R) DNA – deoxyribose (dR)

Nucleobase structures

HypoxanthineInosineInosinate (IMP) XanthineXanthosine Xanthylate (XMP)

Two major routes for nucleotide biosynthesis dNTPs Stryer Fig. 25.1

Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via salvage pathways 1 and 2 (red) 12

Adenine + PRPP Adenylate + PPi adenine phosphoribosyl transferase Guanine + PRPP Guanylate + PPi hypoxanthine-guanine phosphoribosyl transferase Hypoxanthine + PRPP + PPi Phosphoribosyl transferases involved in salvage pathway convert free bases to nucleotides (HGPRT) Inosinate

Biodegradation of Nucleotides (Stryer p )

Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via salvage pathways 1 and 2 (red) 12

Purine biodegradation in humans leads to uric acid

AMP is deaminated to IMP AMP deaminase

IMP is deribosylated to hypoxanthine phosphorylase

Hypoxanthine is oxidized to xanthine

Guanine can be deaminated to give xanthine

Uric acid is the final product of purine degradation in mammals

Uric acid is excreted as urate

Deleterious consequences of defective purine metabolism Gout (excess accumulation of uric acid) Lesch-Nyhan syndrome (HGPRT null) Immunodeficiency

Gout Precipitation and deposition of uric acid causes arthritic pain and kidney stones Causes: impaired excretion of uric acid and deficiencies in HGPRT

Lesch-Nyhan Syndrome Caused by a severe deficiency in HGPRT activity Symptoms are gouty arthritis due to uric acid accumulation and severe neurological malfunctions including mental retardation, aggressiveness, and self-mutilation Sex-linked trait occurring mostly in males

Guanine + PRPP Guanylate + PPi hypoxanthine-guanine phosphoribosyl transferase Hypoxanthine + PRPPInosinate + PPi Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of PRPP, which activates the synthesis of purine nucleotides Excessive uric acid forms as a degradation product of purine nucleotides Basis of neurological aberrations is unknown

Immunodeficiency induced by Adenosine Deaminase defects Defects in AMP deaminase prevent biodegradation of AMP AMP is converted into dATP dATP inhibits the synthesis of deoxyribonucleotides by ribonucleotide reductase, causing problems with the immune system (death of lymphocytes, immunodeficiency disease) AMP deaminase

Summary: Nucleotides have many important functions in a cell. Two major sources of nucleotides are salvage pathway and de novo biosynthesis Purine nucleotides are biodegraded by nucleotidases, nucleotide phosphorylases, deaminases, and xanthine oxidase. Uric acid is the final product of purine biodegradation in mammals Defective purine metabolism leads to clinical disease.

Key concepts in Biosynthesis: Review Committed step Regulated step Allosteric inhibitor Feedback inhibition

De novo Biosynthesis of Pyrimidines Required reading: Stryer’s Biochemistry 5 th Ed., p ,

De novo Biosynthesis of Pyrimidines dTTP Stryer Fig. 25.2

Part 1. The formation of carbamoyl phosphate Enzyme: carbamoyl phosphate synthetase II (CPS) This is the regulated step in pyrimidine biosynthesis

Bicarbonate is phosphorylated CPS

Phosphate is displaced by ammonia: : General strategy for making C-N bonds: C-OH is phosphorylated to generate a good leaving group (phosphate) CPS

General Mechanism for making C-N bonds:

Ammonia necessary for the formation of carbamic acid originates from glutamine:

Structure of Carbamoyl phosphate synthetase II Stryer Fig. 25.3

The active site for glutamine hydrolysis to ammonia contains a catalytic dyad of Cys and His residues Stryer Fig. 25.4

Carbamic acid is phosphorylated CPS

Substrate channeling in CPS Stryer Fig. 25.5

Carbamoyl phosphate supplies the C-2 and the N-3 of the pyrimidine ring dTTP

Part 2. The formation of orotate.

Aspartate is coupled to carbamoyl phosphate Enzyme: aspartate transcarbamoylase This is the committed step in pyrimidine biosynthesis

Stryer Fig Aspartate transcarbamoylase is allosterically inhibited by CTP

Allosteric regulation of Aspartate Transcarbamoylase Stryer Fig. 10.5

PALA is a bisubstrate analog that mimics the reaction intermediate on the way to carbamoyl aspartate Bisubstrate analog

PALA binds to the active site within catalytic subunit Stryer Fig. 10.7

Substrate binding to Aspartate Transcabamoylase induces a large change in ATC quaternary structure Stryer Fig. 10.8

CTP binding prevents ATC transition to the active R state Stryer Fig. 10.9

Allosteric regulation of Aspartate Transcabamoylase Stryer Fig

N-Carbamoylaspartate cyclizes to dihydroorotate - H 2 O

Dihydroorotate dehydrogenase Dihydroorotate is oxidized to orotate

Part 3. The formation of UMP a. Orotate is phosphoribosylated to OMP Pyrimidine phosphoribosyl transferase

b. OMP is decarboxylated to form UMP (OMP) (UMP) OMP decarboxylase (UMP synthetase) Note: phosphoribosyl transfer and decarboxylase activities are co-localized in UMP synthetase

c.Phosphorylation of UMP gives rise to UDP and UTP:

CTP is produced by replacing the 4-keto group of UTP with NH 2 Note: TTP for DNA synthesis is produced via methylation of CTP (will discuss later) CTP synthetase

Regulation of pyrimidine nucleotide biosynthesis OMP decarboxylase (UMP synthetase) Carbamoyl phosphate synthetase CTP synthetase Aspartate transcarbamoylase Regulated step Committed step

Defects in de novo pyrimidine biosynthesis lead to clinical disease Orotic acidurea –Symptoms: anemia, growth retardation, orotic acid excretion –Causes: a defect in phosphoribosyl transferase or orotidine decarboxylase –Treatment: patients are fed uridine U  UMP  UDP  UTP UTP inhibits carbamoyl phosphate synthase II, preventing the biosynthesis and accumulation of orotic acid

PRPP OMP UMP UDP UTP CTP Glutamine + HCO ATP Carbamoyl phosphate Carbamoyl aspartate Carbamoyl phosphate synthetase

Drug inhibitors of pyrimidine biosynthesis Inhibitors of PRPP synthetase:

Inhibitors of dihydroorotase

Pyrimidine biosynthesis: take home message 1.Pyrimidines are synthesized by de novo and salvage pathways. 2. The pyrimidine ring is synthesized from pre-assembled ingredients (carbamoyl phosphate and aspartate) and then attached to the ribose. 3. Pyrimidine biosynthesis is tightly regulated via feedback inhibition (CTP synthetase, carbamoyl phosphate synthetase, aspartate transcarbamoylase) and transcriptional regulation (ATCase). 4. The mammalian enzymes are multifunctional (e.g. carbamoyl phosphate synthetase, UMP synthetase) and form multienzyme complexes to increase efficiency. 5. Drug inhibitors of pyrimidine biosynthesis are under development as potential antimicrobial and anticancer agents.