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Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase
Biochemistry
|June 12, 1979
Summary
Methotrexate slowly inhibits dihydrofolate reductase by forming a complex that isomerizes. This tight binding, with an overall inhibition constant of 58 pM, suggests methotrexate acts as a pseudosubstrate.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Molecular biology
Background:
- Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism.
- Methotrexate is a known inhibitor of DHFR, used in cancer chemotherapy.
- Understanding the precise mechanism of DHFR inhibition is vital for drug development.
Purpose of the Study:
- To elucidate the slow, tight-binding inhibition mechanism of methotrexate on DHFR from Streptococcus faecium.
- To quantify the kinetic parameters governing the interaction between DHFR, methotrexate, and NADPH/NADP.
- To investigate the role of cofactor binding in modulating methotrexate-enzyme interactions.
Main Methods:
- Quantitative analysis of enzyme progress curves.
- Fluorescence quenching assays to monitor complex formation and determine dissociation constants.
- Kinetic modeling to determine inhibition constants and rate constants for isomerization.
Main Results:
- Enzyme kinetics data support a mechanism involving rapid formation of an enzyme-NADPH-methotrexate complex followed by slow isomerization.
- The overall inhibition constant (Ki) for methotrexate was determined to be 58 pM.
- Fluorescence quenching confirmed enzyme-methotrexate complex formation (Kd = 0.36 μM) and showed enhanced binding in the presence of NADPH or NADP.
Conclusions:
- Methotrexate exhibits a slow, tight-binding inhibition of Streptococcus faecium DHFR via a multi-step mechanism.
- Cofactor binding (NADPH/NADP) significantly enhances methotrexate's affinity for DHFR.
- Methotrexate is proposed to function as a pseudosubstrate for dihydrofolate reductase.