The u.v. difference spectra generated when methotrexate, trimethoprim or folate bind to Lactobacillus casei dihydrofolate reductase were analysed. The difference spectrum produced by methotrexate binding is shown to consist of three components: (a) one closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; (b) a pH-independent contribution corresponding to a 40nm shift to longer wavelengths of a single absorption band of methotrexate; (c) a component arising from perturbation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of component (a) shows that the pK of methotrexate is increased from 5.35 to 8.55 (±0.10) on binding. In contrast, folate is not protonated when bound to the enzyme at neutral pH. At pH 7.5, where methotrexate is bound 2000 times more tightly than folate, one-third of the difference in binding energy between the two compounds arises from the difference in charge state. A similar analysis of the difference spectra generated on trimethoprim binding demonstrates that this compound, too, shows an increase in pK on binding, but only from 7.22 to 7.90 (±0.10), suggesting that its 2,4-diaminopyrimidine ring does not bind to the enzyme in precisely the same way as the corresponding moiety of methotrexate.
Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.
568th MEETING, ABERDEEN 77 1 is strictly non-competitive with 3-phospho-~-glycerate at the catalytic centre and strictly competitive with the corresponding MgATP2-. Analogous product-inhibition patterns were previously obtained with ADP, being non-competitive with MgATPZ-and competitive with 3-phospho-~-glycerate (Larsson-Rainikiewicz & Arvidsson, 1971). ADP and 1,3-diphospho-~-glycerate appear preferentially to bind to sites that are involved in the substrate activation observed at elevated concentrations of MgATP2-or 3-phospho-D-glycerate. The kinetic properties of the enzyme offer possibilities for the substrates, even at 'low' concentrations, to control very effectively the direction of the reversible reaction under, for example, conditions in vivo.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is of considerable pharmacological interest as the site of action of the 'anti-folates', a group together with a comparison of the spectra of the complexes with those of the various ionic forms of the substrates, leads to the conclusion that both these substrates bind in their neutral form. It is clear that the pK (for protonation at N-1) of the substrates is not increased on binding as much as that of the inhibitors, indicating some difference in the mode of interaction with the enzyme.
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