Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase

Abstract

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.

Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate

“…In contrast, direct interaction between the side-chain carboxylate of E30 in rhDHFR or D27 in ecDHFR and either N 5 or N 8 of substrate might instead result in a stable charge-mediated hydrogen bond, thus interfering with charge delocalization to the adjacent carbon atom. Just such a strong hydrogen bond is seen in complexes of the tightbinding inhibitor methotrexate with clDHFR (Subramanian & Kaufman, 1978), bovine DHFR (Cocco et al, 1983), ecDHFR (Schlegel et al, 1981;Bolin et al, 1982), and lcDHFR (Hood & Roberts, 1978;Cocco et al, 1981;Bolin et al, 1982). To study the role of the carboxylic acid group, a site-directed mutant of ecDHFR was constructed, replacing D27 with asparagine (D27N) .…”

“…That a similarity between the pH difference spectrum in solution and the binding difference spectrum indicates proton uptake by a ligand has been demonstrated by calorimetric measurements on methotrexate binding to clDHFR (Subramanian & Kaufman, 1978). In contrast, there is no such evidence for protonation when folate binds to rhDHFR (spectra not shown), clDHFR (Subramanian & Kaufman, 1978), ecDHFR (Poe et al, 1974), lcDHFR (Hood & Roberts, 1978), or bacteriophage T4 specific DHFR (Erickson & Mathews, 1972). Moreover, a semiempirical calculation of the heats of formation of both N 1 and N8 protonated 5-deaza-6-methylpteridine (an analogue of 5-deazafolate lacking the pABG moiety) by the AM1 method (Dewar et al, 1985) revealed that N8 protonation is more favorable by 3.9 kcal mol-' (Terry Jones, personal communication).…”

Probing the salt bridge in the dihydrofolate reductase-methotrexate complex by using the coordinate-coupled free-energy perturbation method.

“…Several investigators (5,7,27) have suggested that the strong interaction between MTX and DHFR is the result of the ionic reaction between the protonated MTX and the negatively charged Asp-27 in a hydrophobic environment. However, unless the charged group is stabilized by the protein surroundings, the energy gained by the ionic interactions would be lost for desolvating the charged groups.…”

“…If the total energy is written as Etot = Ebadh + Eele + EvdW, [5] where Ebadh represents the bond, the angle, and the dihedral AGAB = AGAA' + AGA'B- [7] In state A' the solute has the same bond, angle, dihedral, angle, and dihedral terms. This type of decomposition has been used, in earlier studies (25) on solvation of amino acid side chains, to overcome some sampling difficulties during the conversion of a polar solute to a nonpolar solute in water.…”

“…Since Baker (1) first proposed that the tight binding of the 2,4-diamino heterocyclic inhibitors of dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), such as methotrexate (MTX), is due to the increased basicity of the pteridine ring in the inhibitor as compared to the substrate folate, extensive experimental work has been done to substantiate his hypothesis (2)(3)(4)(5)(6)(7)(8). The crystallographic study (9) on the Escherichia coli enzyme-MTX complex clearly indicated the existence of interaction between N-1 atom of MTX and the side-chain carboxyl group of Asp-27.…”