Biosynthesis of Nucleotides

Biosynthesis of Nucleotides

Nucleotides actively participate in many biochemical reactions:

Nucleotides actively participate in many biochemical reactions:

• ATP and GTP as energy sources• ATP and GTP as energy sources

• Uridine derivatives of sugars participate in carbohydrate metabolism

• Uridine derivatives of sugars participate in carbohydrate metabolism

• Coenzymes (NAD, FAD, CoA) are nucleotide derivatives

• Coenzymes (NAD, FAD, CoA) are nucleotide derivatives

• [ATP], [ADP], [AMP] act as allosteric regulators of key enzymes

• [ATP], [ADP], [AMP] act as allosteric regulators of key enzymes

• Monomeric units of nucleic acids• Monomeric units of nucleic acids

Two Pathways for Nucleotide Biosynthesis:Two Pathways for Nucleotide Biosynthesis:

de novo pathway (anew; from scratch): nucleotides are constructed from simple precursors

de novo pathway (anew; from scratch): nucleotides are constructed from simple precursors

salvage pathways: recovery and recycling of nucleotides obtained in the dietsalvage pathways: recovery and recycling of nucleotides obtained in the diet

De Novo Biosynthesis of Purines:De Novo Biosynthesis of Purines:

Studied first in pigeons. Birds excrete nitrogen as the purine uric acid:Studied first in pigeons. Birds excrete nitrogen as the purine uric acid:

NNHH

OONN

NN

NNHH

HH

OO

OO

HH

Pigeons were fed isotopically labeled compounds and the distribution of labeled atoms examined in uric acid.

Pigeons were fed isotopically labeled compounds and the distribution of labeled atoms examined in uric acid.

NN

NN

NN

NN

GlycineGlycine

GlutamineGlutamine

N10-formyl-THFN10-formyl-THF

CO2CO2

AspartateAspartate

N10-formyl-THFN10-formyl-THF

11

99

88

66

55

44

3322

77

Origin of ring atoms of purines:Origin of ring atoms of purines:

Atoms forming the purine ring are successively added to ribose-5-P.Atoms forming the purine ring are successively added to ribose-5-P.

Purines are thus directly synthesized as nucleotide derivatives by assembling the atoms comprising the purine ring directly on ribose.

Purines are thus directly synthesized as nucleotide derivatives by assembling the atoms comprising the purine ring directly on ribose.

Phosphoribosylpyrophosphate (PRPP) is formed from ribose-5-P and ATP by PRPP synthetase. PRPP is the donor of the ribose ring of the nucleotides.

Phosphoribosylpyrophosphate (PRPP) is formed from ribose-5-P and ATP by PRPP synthetase. PRPP is the donor of the ribose ring of the nucleotides.

PRPPPRPP

PRPP also participates in pyrimidine biosynthesis and in the synthesis of histidine and tryptophan.

PRPP also participates in pyrimidine biosynthesis and in the synthesis of histidine and tryptophan.

NH2NH2

NHNHO=CO=C

CH2CH2

NH2NH2

CHCH

OO

NHNHHN=CHN=C

CH2CH2

NHNHCHCH

OO

NNCC

HCHCNN

CHCH

H2NH2N

NNCCCC

NN

CHCH

H2NH2N

-OOC-OOC

NNCCCC

NN

CHCH

H2NH2N

CC

OO

HC-NHHC-NH

CH2CH2

COO-COO-

COO-COO-

NNCCCC

NN

CHCH

H2NH2N

CC

OO

H2NH2N

NNCCCC

NN

CHCH

NHNH

CC

OO

H2NH2N

O=CH-O=CH-

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

HCHC

Inosine Monophosphate (IMP)Inosine Monophosphate (IMP)

AMP and GMP are synthesized from IMPAMP and GMP are synthesized from IMP

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

HCHC

IMPIMP

aspartateGTP

aspartateGTP

Adenylosuccinate Synthetase

Adenylosuccinate Synthetase

GDP, PiGDP, Pi

NNCCCC

NN

CHCH

NN

CCNN

HCHC

NHNH

-OOC-CH2-CH-COO--OOC-CH2-CH-COO-

AdenylosuccinateAdenylosuccinate

NNCCCC

NN

CHCH

NN

CCNN

HCHC

NHNH

-OOC-CH2-CH-COO--OOC-CH2-CH-COO- fumaratefumarate

AdenylosuccinaseAdenylosuccinase

NNCCCC

NN

CHCH

NN

CCNN

HCHC

NH2NH2

AMPAMP

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

HCHC

IMPIMP

NAD+NAD+

IMP D’haseIMP D’hase

NADHNADH

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

O=CO=C

HH

Xanthosine monophosphate

(XMP)

Xanthosine monophosphate

(XMP)

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

O=CO=C

HH

XMPXMP

GMP Synthetase

GMP Synthetase

ATPATP

AMP, PPiAMP, PPiGlutamineGlutamine

GlutamateGlutamate

NNCCCC

NN

CHCH

NN

CC

OO

HNHN

H2N-CH2N-C

GMPGMP

Regulation of Purine Biosynthesis:Regulation of Purine Biosynthesis:

• PRPP synthetase is feedback inhibited by AMP, GMP and IMP.

• PRPP synthetase is feedback inhibited by AMP, GMP and IMP.

• Adenylosuccinate synthetase is inhibted by AMP.

• Adenylosuccinate synthetase is inhibted by AMP.

• IMP d’hase is inhibited by XMP and GMP.• IMP d’hase is inhibited by XMP and GMP.

Purine Salvage:Purine Salvage:

• During cellular metabolism and during digestion in animals, nucleic acids are degraded to mononucleotides, nucleosides, and free purine bases.

• During cellular metabolism and during digestion in animals, nucleic acids are degraded to mononucleotides, nucleosides, and free purine bases.

• Some purines are further degraded to uric acid, but a considerable fraction are directly converted back to purine ribonucleotides

• Some purines are further degraded to uric acid, but a considerable fraction are directly converted back to purine ribonucleotides

AMPAMP

AdenosineAdenosine

AdenineAdeninePRPPPRPP

PPiPPi

Adenine phosphoribosyl transferase

Adenine phosphoribosyl transferase

InosineInosine

HypoxanthineHypoxanthine

Hypoxanthine-guaninephosphoribosyl-

transferase

Hypoxanthine-guaninephosphoribosyl-

transferase

PRPPPRPP

PPiPPiGMPGMP

GuanosineGuanosine

GuanineGuanine

IMPIMP

Lesch-Nyhan Syndrome:Lesch-Nyhan Syndrome:

• Described by William Nyhan and Michael Lesch in 1964.

• Described by William Nyhan and Michael Lesch in 1964.

• Hereditary deficiency of hypoxanthine-guanine phosphoribosyltransferase. Disease affects mostly males.

• Hereditary deficiency of hypoxanthine-guanine phosphoribosyltransferase. Disease affects mostly males.

• Hypoxanthine and guanine are degraded to uric acid instead of being converted to IMP and GMP.

• Hypoxanthine and guanine are degraded to uric acid instead of being converted to IMP and GMP.

• Symptoms: mental retardation; spasticity; bizarre tendency to self-mutilate.

• Symptoms: mental retardation; spasticity; bizarre tendency to self-mutilate.

Pyrimidine BiosynthesisPyrimidine Biosynthesis

• The common pyrimidine ribonucleotides are cytidine-5’-monophosphate and uridine-5’-monophosphate

• The common pyrimidine ribonucleotides are cytidine-5’-monophosphate and uridine-5’-monophosphate

• The pyrimidine ring is synthesized first, then attached to ribose-5-phosphate

• The pyrimidine ring is synthesized first, then attached to ribose-5-phosphate

• Pyrimidine nucleotides are made from aspartate, PRPP and carbamoyl phosphate

• Pyrimidine nucleotides are made from aspartate, PRPP and carbamoyl phosphate

Origin of ring atoms of pyrimidines:Origin of ring atoms of pyrimidines:

CC

NN

CC

CCCC

NN

11

33

22

44

66

55 AspartateAspartateCarbamoyl PhosphateCarbamoyl Phosphate

(N3 originally

from glutamine; C

2 from HCO

3-)

(N3 originally

from glutamine; C

2 from HCO

3-)

GlutamineGlutamine ++ HCO3

HCO3 ++ 2 ATP2 ATP

Carbamoyl Phosphate Synthase II

Carbamoyl Phosphate Synthase II

(committed step in mammals)(committed step in mammals)

H2N-C-OPO

32-H

2N-C-OPO

32-

OO++ 2 ADP, Pi2 ADP, Pi

Carbamoyl PhosphateCarbamoyl Phosphate

Aspartate + carbamoyl phosphateAspartate + carbamoyl phosphate

Aspartate transcarbamoylaseAspartate transcarbamoylase

CC =O=O-O --O -

H2NH2N

CH-COO-CH-COO-

CH2CH2

NNHH

O=CO=C

N-carbamoylaspartateN-carbamoylaspartate

*ATCase was the first allosteric enzyme to be

characterized

*ATCase was the first allosteric enzyme to be

characterized

DihydroorotaseDihydroorotase

H2OH2O

CH-COO-CH-COO-

CCOO

HNHN CH2CH2

NNHH

O=CO=C

L-DihydroorotateL-Dihydroorotate

Dihydroorotate D’hase

Dihydroorotate D’hase

NADHNADH

NAD+NAD+ (Q)(Q)

(QH2)(QH2)

C-COO-C-COO-

CCOO

HNHN CHCH

NNHH

O=CO=C

OrotateOrotate

Orotate

Phosphoribosyl

Transferase

Orotate

Phosphoribosyl

Transferase

PPiPPi

PRPPPRPP

C-COO-C-COO-

CCOO

HNHN CHCH

NNO=CO=C

OrotidylateOrotidylate

Orotidylate Decarboxylase

Orotidylate Decarboxylase

CO2CO2

CHCH

CCOO

HNHN CHCH

NNO=CO=C

Uridine 5’-monophosphate (UMP)

Uridine 5’-monophosphate (UMP)

KinasesKinases

2 ADP2 ADP

2 ATP2 ATP

CHCH

CCOO

HNHN CHCH

NNO=CO=C

Uridine 5’-Triphosphate (UTP)Uridine 5’-Triphosphate (UTP)

Cytidylate SynthetaseCytidylate Synthetase

*Allosterically inhibited by CTP

*Allosterically inhibited by CTP

GlutamateGlutamate

GlutamineGlutamine

CHCH

CCNH2NH2

NN CHCH

NNO=CO=C

Cytidine 5’-Triphosphate (CTP)Cytidine 5’-Triphosphate (CTP)

Mammalian pyrimidine synthesis is an example of metabolite channeling.Mammalian pyrimidine synthesis is an example of metabolite channeling.

In bacteria, the six enzymes of de novo pyrimidine synthesis are separate proteins.In bacteria, the six enzymes of de novo pyrimidine synthesis are separate proteins.

In mammals, the six activities are contained within three proteins. In mammals, the six activities are contained within three proteins.

CPS-II, asparate transcarbamoylase, dihydroorotaseare all contained within a single cytosolic protein. DHO d’hase is localized in the inner mito. membrane. Orotate phosphoribosyltransferase and OMP decarboxylase are contained with a single protein called OMP synthase.

CPS-II, asparate transcarbamoylase, dihydroorotaseare all contained within a single cytosolic protein. DHO d’hase is localized in the inner mito. membrane. Orotate phosphoribosyltransferase and OMP decarboxylase are contained with a single protein called OMP synthase.

Regulation of Pyrimidine Biosynthesis:Regulation of Pyrimidine Biosynthesis:

• Carbamoyl phosphate synthetase II is allosterically activated by PRPP and ATP. Pyrimidine nucleotides (UDP, UTP) inhibit.

• Carbamoyl phosphate synthetase II is allosterically activated by PRPP and ATP. Pyrimidine nucleotides (UDP, UTP) inhibit.

• Aspartate transcarbamoylase (ATCase) from E. coli is inhibited by pyrimidine nucleotides (CTP and UTP). ATP is an allosteric activator.

• Aspartate transcarbamoylase (ATCase) from E. coli is inhibited by pyrimidine nucleotides (CTP and UTP). ATP is an allosteric activator.

• Deoxyribonucleotides are synthesized by reduction of ribonucleosides

• Deoxyribonucleotides are synthesized by reduction of ribonucleosides

• All 4 ribonucleoside diphosphates (ADP, GDP, CDP, UDP) are substrates for Ribonucleotide Reductase

• All 4 ribonucleoside diphosphates (ADP, GDP, CDP, UDP) are substrates for Ribonucleotide Reductase

• Ribonucleotide Reductase has both a catalytic site and two allosteric sites. One allosteric site (Activity site) controls activity at the catalytic site. The second (Specificity site) determines which nucleoside diphosphate binds the active site.

• Ribonucleotide Reductase has both a catalytic site and two allosteric sites. One allosteric site (Activity site) controls activity at the catalytic site. The second (Specificity site) determines which nucleoside diphosphate binds the active site.

Ligand bound to Activity SiteLigand bound to Activity Site

Ligand bound to Specificity SiteLigand bound to Specificity Site

Activity of Catalytic SiteActivity of Catalytic Site

dATPdATP Enzyme InactiveEnzyme Inactive

ATPATP ATP or dATPATP or dATP CDP or UDPCDP or UDP

ATPATP dTTPdTTP GDPGDP

ATPATP dGTPdGTP ADPADP

dTMP is formed from dUMP:dTMP is formed from dUMP:

UMPUMP

Nucleoside Monophosphate Kinase

Nucleoside Monophosphate Kinase

dUMPdUMPUDPUDP

Nucleoside Diphosphate Kinase

Nucleoside Diphosphate Kinase

dUDPdUDP

Ribonucleotide ReductaseRibonucleotide Reductase

dUTPdUTP

dUTPasedUTPase

dTMPdTMPThymidylate SynthaseThymidylate Synthase

dTMP is also formed from dCDP:dTMP is also formed from dCDP:

Cytidine

deaminase

Cytidine

deaminase

dCDPdCDP dCMPdCMP dUMPdUMP

(activated by dCTP

inhibited by dTTP)

(activated by dCTP

inhibited by dTTP)

Of the 4 dNTPs, only dCTP does not interact with the regulatory sites on ribonucleotide reductase, instead it interacts with dCMP deaminase.

Of the 4 dNTPs, only dCTP does not interact with the regulatory sites on ribonucleotide reductase, instead it interacts with dCMP deaminase.

dUMPdUMPCHCH

CCOO

HNHN CHCH

NNO=CO=C

N5N10-Methylene-

tetrahydrofolate

N5N10-Methylene-

tetrahydrofolate

DihydrofolateDihydrofolate

TetrahydrofolateTetrahydrofolate

CHCH

CCOO

HNHN C-CH3C-CH3

NNO=CO=C

dTMPdTMP

Dihydrofolate ReductaseDihydrofolate Reductase

methotrexatemethotrexate

XX

dTMP can also be synthesized via salvage of thymidine:dTMP can also be synthesized via salvage of thymidine:

Thymidine Thymidine Thymidine

kinaseThymidine

kinase

ATPATP ADPADPdTMPdTMP

Radioactive thymidine is used for monitoring intracellular synthesis of DNA because it enters cells easily and its principle metabolic fate is salvage leading to incorporation into DNA.

Radioactive thymidine is used for monitoring intracellular synthesis of DNA because it enters cells easily and its principle metabolic fate is salvage leading to incorporation into DNA.

• Many anticancer drugs target DNA synthesis; particularly thymidylate synthesis

• Many anticancer drugs target DNA synthesis; particularly thymidylate synthesis

• Methotrexate and aminopterin inhibit dihydrofolate reductase; thymidylate cannot be formed thus DNA cannot be replicated

• Methotrexate and aminopterin inhibit dihydrofolate reductase; thymidylate cannot be formed thus DNA cannot be replicated

• 5-Fluorouracil is converted to 5-Fluorodeoxy- uridylate which binds tightly to thymidylase synthase and inhibits the enzyme.

• 5-Fluorouracil is converted to 5-Fluorodeoxy- uridylate which binds tightly to thymidylase synthase and inhibits the enzyme.

• Azaserine and acivicin are glutamine analogues also used as chemotheraputic agents

• Azaserine and acivicin are glutamine analogues also used as chemotheraputic agents