Biosynthesis of nucleotides

Biosynthesis of nucleotides

Natalia Tretyakova, Ph.D.

Phar 6152

Spring 2004

Required reading: Stryer’s Biochemistry 5th edition, p. 262-268, 693-712

(or Stryer’s Biochemistry 4th edition p. 238-244, 739-759)

Tentative Lecture plan: Biosynthesis of Nucleotides

03-31 Introduction. Biological functions and sources of nucleotides. Nucleotide metabolism.

04-02 Biosynthesis of pyrimidine ribonucleotides.

04-05 Biosynthesis of purine ribonucleotides

04-07 Biosynthesis of deoxyribonucleotides. Inhibitors of nucleotide metabolism as drugs.

04-09 Review

04-12 Exam

Biological functions and sources of nucleotides.

Nucleotide metabolism

Required reading: Stryer’s Biochemistry 5th Ed., p. 693-694, 709-711

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

-OO

H(OH)

HH

HHO

OP

O

O-

Purine orPyrimidineBase

Phosphate

Pentose sugar

Nucleoside

Nucleotide

1'

2'3'

4'

5'

Nucleotides

-glycosidic bond

RNA- ribose (R)DNA – deoxyribose (dR)

NN

NNH

NH2

NNH

NH

NH

O

O

N

NH

NH2

O

H3C

NH

NH

O

O

XanthineAdenine (A)

Thymine (T)Cytosine (C)

NH

NH

O

O

Uracil (U)

NN

NNH

Purine

N

NH

Pyrimidine

1

2

3

4

5

6

3

2

16

4

57

8

9

NNH

NNH

O

NH2

Guanine (G)

NNH

NNH

O

Hypoxanthine

Nucleobase structures

Hypoxanthine Inosine Inosinate (IMP)Xanthine Xanthosine Xanthylate (XMP)

Two major routes for nucleotide biosynthesis

dNTPs

dNTPs

Stryer Fig. 25.1

Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via

salvage pathways 1 and 2 (red)

1 2

Adenine + PRPP Adenylate + PPi

adenine phosphoribosyl transferase

Guanine + PRPP Guanylate + PPi

hypoxanthine-guanine phosphoribosyl transferase

Hypoxanthine + PRPP + PPi

O

OHOH

CH2

OP

OP

O-O

-OO-

2- O3PO

O

5-phosphoribosyl-1-pyrophosphate (PRPP)

Base +

Phosphoribosyl transferases involved in salvage pathway convert free bases to nucleotides

(HGPRT)

Inosinate

O

OHOH

CH2

2- O3PO Base

+ PPi

Biodegradation of Nucleotides

Base

2-O3PO

O

OHOH

HH

HH

NucleootidaseBase

HO

O

OHOH

HH

HH

Base

HO

O

OHOH

HH

H OPO32-

Salvage Degradation

2-O3PO

O

OHOH

HH

H

Nucleotide 5'-phosphate Nucleoside

Ribose-1-phosphate

Ribose-5-phosphate

nucleoside phosphorylase

phosphoribomutase

PRPP

OH

5'

(Stryer p. 709-711)

Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via

salvage pathways 1 and 2 (red)

1 2

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

NH

NNH

N

O

Hypoxanthine

NH

NNH

N

O

Allopurinol

NH

NH

NH

N

O

Alloxanthine

O

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 + PRPP Inosinate + 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)

AMPdeaminase

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 clinicaldisease.

Key concepts in Biosynthesis: Review

•Committed step

•Regulated step

•Allosteric inhibitor

•Feedback inhibition

De novo Biosynthesis of Pyrimidines

Required reading: Stryer’s Biochemistry 5th Ed., p. 262-267, 694-698

-O

N

NH2

ON

O

OHOH

HH

HH

OP

O-

O

-O

NH

O

ON

O

OHOH

HH

HH

OP

O-

O

-O

NH

O

ON

O

HOH

HH

HH

OP

O-

O

De novo Biosynthesis of Pyrimidines

NH2

CO O

PO3-H2N

CH

COO-

CH2

C-O

O

N

NH

NH2

O

CH3HN

NH

O

O

Thymine (T) Cytosine (C)

HN

NH

O

O

Uracil (U)

O

OHOH

CH2

OP

OP

O-O -O O-

2- O3PO

O

dTTPStryer Fig. 25.2

Part 1. The formation of carbamoyl phosphate

OC

NH2

O

PO

-O

O-

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 isphosphorylated to generate a good leaving group (phosphate)

CPS

General Mechanism for making C-N bonds:  

O

R

R'

OH

R

R'

ATP ADP

O

R

R'

P

O-

O-

O

NH3

O

R

R'

P

O-

O-

O

NH2

Pi

NH2

R

R'

NH2

CHCC

H2

O-

O

H2C

C

H2N

O

CPSNH3

Glutamine (Gln) Glutamate

NH2

CHCC

H2

O-

O

H2C

C

-O

O

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

NH2

CHCC

H2

O-

O

H2C

C

H2N

O

CPSNH3

Glutamine (Gln) Glutamate

NH2

CHCC

H2

O-

O

H2C

C

-O

O

Stryer Fig. 25.4

Carbamic acid is phosphorylated

CPS

Substrate channeling in CPS

NH2

CHCC

H2

O-

O

H2C

C

H2N

O

CPSNH3

Glutamine (Gln) Glutamate

NH2

CHCC

H2

O-

O

H2C

C

-O

O

Stryer Fig. 25.5

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

NH2

CO O

PO3-H2N

CH

COO-

CH2

C-O

O

dTTP

Part 2. The formation of orotate.

Aspartate is coupled to carbamoyl phosphate

Enzyme: aspartate transcarbamoylase

OC

NH2

O

PO

-OO-

Pi

NH2

CH-OOC

CH2

COO-

CNH2

O

HN

CHOOC

CH2

COO

Carbamoyl phosphateCarbamoylaspartate

Asp replaces Pi

Asp

This is the committed step in pyrimidine biosynthesis

Stryer Fig. 10.2

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. 10.10

N-Carbamoylaspartate cyclizes to dihydroorotate

CN

O

HN

CHOOCCH2

CO-

Tetrahedral transition state

O-

H

H

CNH2

O

HN

CHOOC

CH2

COO

H+H2O C

NH

O

HN

CHOOCCH2

CODihydroorotase

N-Carbamoylaspartate Dihydroorotate

- H2O

Dihydroorotate dehydrogenase

Dihydroorotate is oxidized to orotate

C

HN

O

N C

COO

CH

C

O

O

OHOH

CH2

-O3POO

OHOH

CH2

OP

OP

O-O

-OO-

-O3PO

O

CNH

O

HN

COOCCH

CO

PPi

Orotate

Orotidylate(orotidine monophosphate, OMP)

5-phosphoribosyl-1-pyrophosphate (PRPP)

Part 3. The formation of UMP

a. Orotate is phosphoribosylated to OMP

Pyrimidine phosphoribosyltransferase

O

OHOH

CH2

OH

2- O3POATP

AMP

ribose-5-phosphate(from pentose phosphate pathway)

PRPP synthetase

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:   

UMP + ATP UDP + ADP

UMP kinase

UDP + ATP UTP + ADP

nucleotide diphosphate kinase

CTP is produced by replacing the 4-keto group of UTP with NH2

Note: TTP for DNA synthesis is produced via methylation of CTP (will discuss later)

CTP synthetase

C

N

O

N C

H

CH

C

O

O

OHOH

CH2

4- O3PO3PO3PO-H2C

P O-

O-

O

4

Regulation of pyrimidine nucleotide biosynthesis

PRPP

OMP

UMP

UDP

UTP

CTP

Glutamine + HCO3- + ATP

Carbamoyl phosphate

Carbamoyl aspartate

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 uridineU UMP UDP UTP

UTP inhibits carbamoyl phosphate synthase II, preventing the biosynthesis and accumulation of orotic acid

O

OHOH

CH2

OP

OP

O-O -O O-

-O3PO

O

CNH

O

HN

COOCCH

CO

PPi

Orotate 5-phosphoribosyl-1-pyrophosphate (PRPP)

UTP inhibits carbamoyl phosphate synthase II, preventing the biosynthesis and accumulation of orotic acid

PRPP

OMP

UMP

UDP

UTP

CTP

Glutamine + HCO3- + ATP

Carbamoyl phosphate

Carbamoyl aspartate

Carbamoyl phosphate synthetase

Drug inhibitors of pyrimidine biosynthesis

Inhibitors of PRPP synthetase:

N

NN

N

NHO

OHOH

HH

HH

HO

OCH3

N

NN

N

NHO

OHOH

HH

HH

HO

NH2

MRPP (MP)noncompetitive, Ki = 190 M

ARPP (MP)noncompetitive, Ki = 430 M

O

OHOH

CH2

OH

2- O3PO ATP AMP

ribose-5-phosphate(from pentose phosphate pathway)

PRPP synthetase

O

OHOH

CH2

OP

OP

O-O

-OO-

-O3PO

O

CNH2

O

HN

CHOOC

CH2

COO

CN

O

HN

CHOOCCH2

CO-

H

H

O-

H+ H2O CNH

O

HN

CHOOCCH2

CO

Carbamoylaspartate DihydroorotateTetrahedral Transition State

Inhibitors of dihydroorotase

NHHC

CHOOCNH

CO

HS

PCH

O

HC

CHOOCNH

CO

SH

MOAC

NHHC

CHOOCNH

CO

O-O

HDDP MMDHO

Ki = 0.14 M Ki = 0.74 M Ki = 2.9 M

Pyrimidine biosynthesis: take home message1.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.