Thermodynamic characterization of the interactions of methotrexate with dihydrofolate reductase by quantitative affinity chromatography

Mark C. Waltham, John W. Holland, Peter F. Nixon, Donald J. Winzor

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Abstract

Affinity chromatography on methotrexate-Sepharose has been used to evaluate dissociation constants for interactions of methotrexate with dihydrofolate reductase from Lactobacillus casei. Equilibrium constants of 0.25 μM and 0.6 nM were obtained for dissociation of the inhibitor from the enzyme-methotrexate and enzyme-NADPH-methotrexate complexes, respectively, these estimates being in good agreement with the corresponding published values for dihydrofolate reductase from Streptococcus faecium. By employing a different method for evaluating the thermodynamic dissociation constant for the enzyme-NADPH-methotrexate interaction, this investigation provides independent support for the inference drawn from published fluorescence quenching studies that the interaction of methotrexate with dihydrofolate reductase-NADPH complex is governed by a dissociation constant in the vicinity of 600 pM, a value slightly higher than, but approaching, the inhibition constant of 50-60 pM obtained by enzyme kinetic techniques.

Original languageEnglish
Pages (from-to)541-545
Number of pages5
JournalBiochemical Pharmacology
Volume37
Issue number3
DOIs
Publication statusPublished - 1 Feb 1988
Externally publishedYes

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