Spectral Changes Associated with Binding of Folate Compounds to Bacteriophage T4 Dihydrofolate Reductase

Dihydrofolate reductase from a resistant subline of the L1210 lymphoma. Purification by affinity chromatography and ultraviolet difference spectrophotometric and circular dichroic studies

“…The difference spectra of L1210/MTX reductase obtained upon binding with NADP and NADPH4" are virtually identical with those reported for the first or tighter pyridine nucleotide binding site for the E. coli enzyme (Poe et al, 1974b). However, this is in contrast to the T4-phage reductase (Erickson and Mathews, 1972). In this enzyme, NADPH addition had virtually no effect on the spectrum, in The molar circular dichroism of the reductase and 1:1 complexes with NADP+ and NADPH at 27 °C from 375 to 250 nm: (-)…”

“…The Li 210/MTX enzyme-MTX complex difference spectrum is aiso almost superimposable on the difference spectrum noted for E. coli reductase upon binding of MTX (Poe et al, 1974a) with the exception of the negative band at 315 nm. The origin of this band is unknown; however, it was also observed upon binding of MTX to T4-phage reductase (Erickson and Mathews, 1972). The E. coli and T4 enzyme-MTX difference spectra have been interpreted as preferential binding of the acidic form of MTX (protonated at N-l of the 2,4-diaminopteridine moiety) (Erickson and Mathews, 1972;Poe et al , 1974a) by the enzyme.…”

“…The origin of this band is unknown; however, it was also observed upon binding of MTX to T4-phage reductase (Erickson and Mathews, 1972). The E. coli and T4 enzyme-MTX difference spectra have been interpreted as preferential binding of the acidic form of MTX (protonated at N-l of the 2,4-diaminopteridine moiety) (Erickson and Mathews, 1972;Poe et al , 1974a) by the enzyme. The N-l protonated form of MTX to the E. coli enzyme is almost 105 times smaller than the Nl unprotonated form.…”

Dihydrofolate reductases of Escherichia coli and bacteriophage T4. Spectrofluorometric study

“…Thus, a given salt-induced change in enzyme structure could conceivably alter the binding determinants for dihydrofolate to a greater extent than the binding determinants for the 4-amino compounds. Evidence for this latter explanation is presented elsewhere (Erickson and Mathews, 1972).…”

1 H nuclear magnetic resonance studies of Lactobacillus casei dihydrofolate reductase: effects of substrate and inhibitor binding on the histidine residues

“…Its p if is decreased (by 0.27-0.62 units) on the binding of compounds containing the 2, 4-diaminopyrimidine ring. I t has been proposed (Baker 1959(Baker , 1967 th a t the 4-amino compounds, in contrast to the 4-keto series, bind to the enzyme in the protonated (positively charged) form, and there is some experimental support for this proposal (Baker & Jordaan 1965;Erickson & Mathews 1972;Poe, Greenfield, Hirschfield & Hoogsteen 1974; K. Hood, G .C .K . Roberts & A .S.V .…”