Carbon-13 nuclear magnetic resonance study of protonation of methotrexate and aminopterin bound to dihydrofolate reductase

Abstract

Methotrexate, aminopterin, and folate have been synthesized with 90% enrichment of C-2 with 13C. 13C nuclear magnetic resonance has been used to examine the state of protonation of the pteridine ring of these compounds under various conditions and gives much more clear-cut results than most other methods. For the free compounds the following pK values were obtained: methotrexate, 5.73 +/- 0.02 (N-1); aminopterin, 5.70 +/- 0.03 (N-1); folic acid, 2.40 (N-1) and 8.25 +/- 0.05 (N-3, O-4 amide group). The state of protonation of these compounds when complexed to dihydrofolate reductase (isoenzyme 2 from Streptococcus faecium) was also studied over the pH range 6--10. The resonance from bound methotrexate showed a constant chemical shift over the whole pH range studied, and it is inferred that in the complex the pteridine ring remains protonated to at least pH 10. The same result was obtained for the binary complex of aminopterin with the reductase and for either methotrexate or aminopterin in ternary complex with reductase and NADPH4. The latter is an inhibitor of the reductase competitive with NADPH. However, folate bound to the reductase in either the binary or the ternary complex shows the same protonation behavior as in the free state. The data indicate that the association constant for binding of methotrexate is increased enough when protonation of N-1 occurs to account for the enhanced binding of methotrexate as compared with folate.

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

“…We observe directly a proton at N1 of MTX, imparting it with a positive charge. This result agrees with nearly all available biochemical and complementary structural data for E. coli and L. casei DHFRs: that the MTX⅐D27 interaction is ionic in nature (45)(46)(47)(48)(49)(50)(51). However, it is in disagreement with the theoretical calculations of Cannon et al (52), who proposed that the MTX N1⅐D27 pair involves a neutral dipole-dipole interaction.…”

The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase

“…There appear to be two major differences between the binding of methotrexate and that of folate: methotrexate is protonated at N1 when bound to the enzyme [2][3][4][5][6][7][8][9][10], while folate is apparently not, and the pteridine ring of methotrexate binds to the enzyme in an orientation which differs by a rotation of about 180 ° about its long axis from that adopted by folate [11][12][13][14]. The recent determination of the crystal structure of the enzyme-trimethoprim complex [15] shows that the orientation of the 2,4-diaminopyrimidine ring in the binding site is very similar to that of the analogous part of methotrexate [11,16].…”

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

“…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.…”

“…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.…”

“…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.…”