Dr. Aga Syed SameerLecturer (Demonstrator)

Department of Biochemistry,

Medical College,

Sher-I-Kashmir Institute of Medical Sciences,

Bemina, Srinagar, Kashmir, 190010. India.

Nucleic Acid MetabolismPyrimidine Metabolism

Pyrimidine Biosynthesis

• UMP (Uridine Monophosphate)• Precursor to

• TMP and CTP

• Utilizes (Substrates)• One Aspartate

• γ-NH3 of Glutamine

• CO2 is recycled (Used in 1st step and released back in last)

• Water molecule

• NAD+

• ATP’s (Two : Till UMP Synthesis)

• Methyl group of THF + GTP {dTMP Synthesis}

• NH3 of Glutamine + ATP (2 Equivalent) {CTP Synthesis}

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

C

N

H

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

C

N

H

CH

CH2

C

COOO

O

Dihydroorotate

HN

C

N

H

C

CH

C

COOO

O

Orotate

HN

C

N

C

CH

C

COOO

O

HH

CH2

OH OH

H H

OO

2-O3P

Orotidine-5'-monophosphate

(OMP)

HN

C

N

CH

CH

C

O

O

HH

CH2

OH OH

H H

OO

2-O3P

Uridine Monophosphate

(UMP)

2 ADP +

Glutamate +

Pi

Carbamoyl

Phosphate

Synthetase II

Aspartate

Transcarbamoylase

(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

Reduced

QuinoneDihydroorotate

Dehydrogenase

PRPP PPi

Orotate Phosphoribosyl

Transferase

CO2

OMP

Decarboxylase

Pyrimidine Biosynthesis

Enzymes and Reactions• 2 condensation Rxns: form carbamoyl aspartate and

dihydroorotate (intramolecular)

• Dihydroorotate dehydrogenase (FMN) is an intra-mitochondrial enzyme; oxidizing power comes from ubiquinone reduction

• Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P.

• OMP Decarboxylase enhances the rate of decarboxylation by a factor of 2x1023

• Channeling: • Enzymes 1, 2, and 3 are on same chain;

• Enzymes 5 and 6 are on same chain

CTP and UTP synthesis• UMP is then converted UDP &

UTP

• The conversion takes place by the action of Nucleoside Mono/Di Phosphate Kinases.

• CTP formed from UTP via CTP Synthetase driven by ATP hydrolysis • Glutamine provides amide

nitrogen

Conversion of Ribonucleotidesto Deoxyribonucleotides• Ribonucleotide reductase

• NADP

• Thioredoxin reductase

Regulation of RNR’s

• ATP activates

• dATP Inhibits

dTMP synthesis• dUMP formed; produces dTMP via Thymidylate

Synthetase• N5,N10 Methylene THF provides methyl group

• THF is first reduced and then methylated

dTMP synthesis

Regulation• Differs between bacteria and animals

• Bacteria – regulation at ATCase rxn

• Animals – regulation at carbamoyl phosphate synthetaseII

• UDP and UTP inhibit enzyme;

• ATP and PRPP activate it

• Also UMP and CMP competitively inhibit OMP Decarboxylase

Degradation of Pyrimidines• CMP and UMP degraded to bases similarly to purines

• Dephosphorylation

• Deamination

• Glycosidic bond cleavage

• Uracil reduced in liver, forming β-Alanine

• Is then converted to malonyl-CoA used in fatty acid synthesis for energy metabolism

• dTMP is degraded to β-Amino Isobutyrate

• Is then converted to methyl malonyl-CoA used in fatty acid synthesis for energy metabolism

Cancer is a clonal disorder.

It is composed of malignant cells of

several distinguishable characteristics

such as:

immortality,

faster growth,

unable to establish cell-cell

interaction,

propensity to invade, metastasize and

grow in an abnormal cellular

environment.

Hanahan D and Weinberg RA. The hallmarks of cancer. Cell. 2000; 100:57-70.

Cancer

Molecules Implicated in Colorectal Cancer PathogenesisIn Kashmiri Population

Aga Syed Sameer

Anti-Folate Drugs • Cancer cells consume dTMP quickly for DNA

replication

• Interfere with Thymidylate Synthase rxn to decrease dTMP production • (fluorodeoxyuridylate – irreversible inhibitor) – also affects

rapidly growing normal cells (hair follicles, bone marrow, immune system, intestinal mucosa)

• Dihydrofolate reductase step can be stopped competitively (DHF analogs)

• Anti-Folates: Aminopterin, methotrexate, trimethoprim

Thymidylate Synthesis and Cancer Chemotherapy

• Thymidylate synthase is target for fluorouracil• Action is 5-fluorouracil (5-FU)is converted to 5-fluoro-2’-

deoxyuridylate (dUMP structural analog)

• Then 5-fluoro-2’-deoxyuridylate binds to the enzyme Thymidylate Synthase and undergoes a partial reaction where part of the way through 5-fluoro-2’-deoxyuridylate forms a covalent bridge between Thymidylate Synthase and N5, N10-Methylene THF and is an irreversible inhibition.• Normally, the enzyme, Thymidylate Synthase and the vitamin would

NOT be linked together permanently

• This type of inhibition is called “suicide-based enzyme inhibition”

Thymidylate Synthesis and Cancer Chemotherapy

Methotrexate• Competitive inhibitor of Dihydrofolate Reductase

• Used in,

• Acute lymphoblastic leukemia

• Osteosarcoma in children

• Solid tumor treatment

• Breast, head, neck, ovary, and bladder

• Prevents regeneration of tetrahydrofolate and removes activity of the active forms of folate