Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase

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Abstract
Investigations were made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase [EC 1.5.1.3] from Streptococcus faecium A. Quantitative analysis showed that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme[E].sbd.NADPH.sbd.methotrexate complex that subsequently undergoes a relatively slow reversible isomerization reaction. From the Ki [inhibition constant] value for the dissociation of methotrexate from the E.sbd.NADPH.sbd.methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme.sbd.methotrexate complex was demonstrated by fluorescence quenching, and a value of 0.36 .mu.M was determined for its Kd. The same technique was used to determine Kd for the reaction of methotrexate with the E.sbd.NADP and E.sbd.NADPH complexes. In the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. Methotrexate apparently behaves as a pseudosubstrate for dihydrofolate reductase.