Antimetabolite mechanism: Methotrexate is toxic to human cells primarily because it inhibits which folate-dependent step in nucleotide biosynthesis?

Introduction / Context:
Methotrexate is a classic chemotherapeutic and immunosuppressive antimetabolite. Understanding its precise biochemical target explains both its efficacy and toxicity profile in rapidly dividing cells, including bone marrow and gastrointestinal epithelium.

Given Data / Assumptions:

• Methotrexate structurally resembles folate.
• Folate coenzymes (THF forms) donate one-carbon units in purine and thymidylate synthesis.
• Blocking THF regeneration halts DNA synthesis.

Concept / Approach:
Methotrexate competitively inhibits dihydrofolate reductase (DHFR), the enzyme that reduces dihydrofolate (H2 folate) to tetrahydrofolate (H4 folate). Without H4 folate, cells cannot generate 10-formyl-THF and 5,10-methylene-THF required for de novo purine and dTMP synthesis, leading to S-phase arrest and cytotoxicity in proliferating cells.

Step-by-Step Solution:

Verification / Alternative check:
Leucovorin (folinic acid), a reduced folate, rescues normal cells by bypassing DHFR blockade, confirming the site of action.

Why Other Options Are Wrong:

• CTP formation from UTP is catalyzed by CTP synthase, not folate-dependent.
• PRPP synthesis is governed by PRPP synthetase; methotrexate does not inhibit this step.
• Thymidylate synthase uses 5,10-methylene-THF; methotrexate indirectly depletes this cofactor rather than directly inhibiting the enzyme (that is the mechanism of 5-fluorouracil).
• Formylation of Met-tRNAi is a prokaryotic initiation step and unrelated to methotrexate.

Common Pitfalls:
Confusing direct enzyme targets (TS vs DHFR); overlooking that folate antagonism affects both purine and pyrimidine pathways via THF coenzymes.

Final Answer:
Reduction of dihydrofolate (H2 folate) to tetrahydrofolate (H4 folate)