NM2

Purine and Pyrimidine Metabolism 2

Key Concepts

• Thymine Nucleotides:

  • Derived from deoxyuridine monophosphate (dUMP).

• Conversion of Ribonucleotides to Deoxyribonucleotides:

  • Process facilitated by ribonucleotide reductase, a highly regulated enzyme.

• Nucleotide Phosphorylation:

  • Nucleoside diphosphates (NDPs) convert to triphosphates through enzymatic phosphorylation reactions involving nucleoside diphosphate kinase (NDPK).

• One-Carbon Metabolism:

  • Crucial for synthesizing deoxythymidine monophosphate (dTMP).

• Chemotherapeutic Targets:

  • Enzymes in nucleotide biosynthetic pathways are targets for drugs treating cancer and other diseases.

DNA vs RNA

Nucleotide Structure

• Components of Nucleotides:

  • Nitrogenous Base: Attached to a sugar (ribose in RNA and deoxyribose in DNA).

  • Phosphate Groups:

    • Monophosphate, diphosphate, or triphosphate forms.

  • Nitrogenous Bases:

    • Purines: Adenine (A) and Guanine (G)

    • Pyrimidines: Cytosine (C), Thymine (T), and Uracil (U)

Structure Diagrams

• Nitrogenous bases connected by phosphodiester linkages in nucleotide chains.

Overall Scheme of Deoxynucleotide Synthesis

• Conversion Pathways:

  • Ribonucleotides converted successively by:

    • Ribonucleotide reductase (nucleoside diphosphate to deoxynucleoside diphosphate).

    • NDPK (deoxynucleotide diphosphate to triphosphate).

    • Enzymes involved: nucleoside deaminase, reductase, kinase, dUTPase, thymidylate synthase.

Ribonucleotide Reductase Enzyme

Function and Regulation

• Function: Converts ribonucleotides into deoxyribonucleotides.

• Regulatory Sites:

  • Includes primary regulation and substrate specificity sites responsive to allosteric effectors (ATP, dATP, dGTP, dTTP).

• Mechanism:

  • Substrate specificity shifts according to nucleotide concentrations and presence of ATP or dATP.

• Active Site: Contains catalytic subunits that include cofactors like Fe3+ that stabilize reactive radicals in the catalytic process.

Nucleoside-Diphosphate Kinase Mechanism

• Process Overview:

  1. NDPK binds a nucleoside triphosphate (NTP).

  2. Transfers a phosphoryl group to His in the active site.

  3. Forms a phosphoenzyme intermediate, releases bound NDP, and binds a new NDP, completing phosphorylation.

Thymidylate Synthase Mechanism

Enzymatic Action

• Process:

  • Converts dUMP to dTMP utilizing a methyl group from N5,N10-methylene-tetrahydrofolate (THF).

• Role of Folate:

  • Acts as a carbon donor in the synthesis process, with several conversions of folate derivatives leading to active cofactor forms.

Inhibition by Anticancer Drugs

• 5-Fluorouracil (5-FU):

  • Analog of dTMP, irreversibly binds and inhibits thymidylate synthase preventing DNA synthesis.

• Methotrexate:

  • Inhibits dihydrofolate reductase, interferences in THF regeneration which reduces dTMP synthesis leading to reduced DNA synthesis.

• Hydroxyurea:

  • Disrupts ribonucleotide reductase activity, blocking the formation of dNTPs.

Checkpoints for Review

• From which nucleotide are thymine nucleotides derived?

• How are ribonucleotides converted to deoxyribonucleotides?

• Explain the regulation of RNR and its importance to nucleotide balance.

• Describe the ping pong mechanism of NDPK.

• What are one-carbon metabolism reactions and how do they relate to thymidine synthesis?

• Explain the mechanisms of action for hydroxyurea, 5-FU, methotrexate, trimethoprim, and aminopterin.