Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand

Stopped-flow spectrophotometry and stopped-flow fluorometry have been used to study the binding of methotrexate (MTX) and 3-deazamethotrexate (3-deazaMTX) to dihydrofolate reductase (DHFR) isoenzymes from Streptococcus faecium and from Lactobacillus casei. The absorbance change and fluorescence quenching that occur when MTX binds to DHFR isoenzyme II from S. faecium (SFDHFR II) are both biphasic and give similar apparent rate constants for both phases. The faster phase has an apparent rate constant that is dependent on MTX concentration and therefore corresponds to the initial binding reaction. From the concentration dependence it has been calculated that the association rate constant is 3.0 X 10(5) M-1 s-1 at 20 degrees C and pH 7.3, and the association constant (equilibrium constant) under these conditions is 5.8 X 10(5) M-1. By examination of the amplitude of the fast-phase absorbance change at various wavelengths, it has been determined that the absorbance change occurring in the fast phase is due to MTX protonation. Within the limits of the method it was thus not possible to detect a difference in the rates of binding and of protonation of MTX. The MTX association rate constant is pH dependent, decreasing 330-fold as the pH is decreased from 5.0 to 9.0. The data fit well to a curve generated by assuming a single ionization with a pKa of 6.0 and a pH-independent association rate constant 1000-fold greater for binding of protonated MTX to SFDHFR II than for binding of unprotonated MTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility

The partial specific volume, v, and adiabatic compressibility, beta(s), of Escherichia coli dihydrofolate reductase were measured at 30 degrees C in the presence of various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP, methotrexate, and KCl). Binding of these ligands (binary and ternary complexes) brought about large changes of v (0.734-0.754 cm(3) g(-1)) and beta(s) (6. 6x10(-6)-9.8x10(-6) bar(-1)), keeping a linear relationship between the two parameters. The values of v and beta(s) increased with an increase in internal cavity, V(cav), and a decrease in accessible surface area, ASA, which were calculated from the X-ray crystal structures of the complexes. A large variation of V(cav) relative to ASA by ligand binding suggested that the cavity is a dominant factor and the effect of hydration might be small for the ligand-induced changes of v and beta(s). The beta(s) values of the binary and ternary complexes suggested a characteristic conformational flexibility of the kinetic intermediates in the enzyme reaction coordinate. Comparison of beta(s) with the cavity distribution in the crystal structures revealed that the flexibility of the intermediates was mainly determined by the total cavity volume with minor contributions of the number, position, and size of cavities. These results demonstrate that the compressibility is a useful measure of the conformational flexibility of the intermediates in the enzyme reaction and that the combined study of compressibility and X-ray crystallography gives new insight into the protein dynamics through the behavior of the cavities.     

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.     

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

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.     

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase

The binding of trimethoprim and [1,3,2-amino-¹⁵N₃]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by ¹⁵N and ¹H NMR spectroscopy. ¹⁵N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The ¹⁵N chemical shifts and ¹H-¹⁵N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.The N1-proton resonance in the complex has been assigned using the ¹⁵N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s^-1 at 313 K, with an activation energy of 75 (±9) kJ·mole^-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.     

Mass spectrometry on hydrogen/deuterium exchange of dihydrofolate reductase: effects of ligand binding

To address the effects of ligand binding on the structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the hydrogen/deuterium (H/D) exchange kinetics of its binary and ternary complexes formed with various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP(+), and methotrexate) were examined using electrospray ionization mass spectrometry. The kinetic parameters of H/D exchange reactions, which consisted of two phases with fast and slow rates, were sensitively influenced by ligand binding, indicating that changes in the structural fluctuation of the DHFR molecule are associated with the alternating binding and release of the cofactor and substrate. No additivity was observed in the kinetic parameters between a ternary complex and its constitutive binary complexes, indicating that ligand binding cooperatively affects the structural fluctuation of the DHFR molecule via long-range interactions. The local H/D exchange profile of pepsin digestion fragments was determined by matrix-assisted laser desorption/ionization mass spectrometry, and the helix and loop regions that appear to participate in substrate binding, largely fluctuating in the apo-form, are dominantly influenced by ligand binding. These results demonstrate that the structural fluctuation of kinetic intermediates plays an important role in enzyme function, and that mass spectrometry on H/D exchange coupled with ligand binding and protease digestion provide new insight into the structure-fluctuation-function relationship of enzymes.     

Bovine liver dihydrofolate reductase: purification and properties of the enzyme.

A purification procedure is reported for obtaining bovine liver dihydrofolate reductase in high yield and amounts of 100-200 mg. A key step in the procedure is the use of an affinity gel prepared by coupling pteroyl-L-lysine to Sepharose. The purified reductase has a specific activity of about 100 units/mg and is homogeneous as judged by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and titration with methotrexate. The products of the first step of Edman degradation indicated a minimum purity of 79%. The reductase has a molecular weight of about 21500 on the basis of amino acid composition and 22100 +/- 300 from equilibrium sedimentation. It is not inhibited by antiserum to the Streptococcus faecium reductase (isoenzyme 2). Unlike the reductase of many other vertebrate tissues, the bovine enzyme is inhibited by mercurials rather than activated and it has a single pH optimum at both low and high ionic strength. However, the position of the pH optimum is shifted and the activity increased by increasing ionic strength. Automatic Edman degradation has been used to determine 34 of the amino-terminal 37 amino acid residues. Considerable homology exists between this region and the corresponding regions of the reductase from S. faecium and from Escherichia coli. This strengthens the idea that this region contributes to the structure of the binding site for dihydrofolate.     

Role of ionic interactions in ligand binding and catalysis of R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR), which catalyzes the NADPH dependent reduction of dihydrofolate to tetrahydrofolate, belongs to a type II family of R-plasmid encoded DHFRs that confer resistance to the antibacterial drug trimethoprim. Crystal structure data reveals this enzyme is a homotetramer that possesses a single active site pore. Only two charged residues in each monomer are located near the pore, K32 and K33. Site-directed mutants were constructed to probe the role of these residues in ligand binding and/or catalysis. As a result of the 222 symmetry of this enzyme, mutagenesis of one residue results in modification at four related sites. All mutants at K32 affected the quaternary structure, producing an inactive dimer. The K33M mutant shows only a 2-4-fold effect on K(m) values. Salt effects on ligand binding and catalysis for K33M and wildtype R67 DHFRs were investigated to determine if these lysines are involved in forming ionic interactions with the negatively charged substrates, dihydrofolate (overall charge of -2) and NADPH (overall charge of -3). Binding studies indicate that two ionic interactions occur between NADPH and R67 DHFR. In contrast, the binding of folate, a poor substrate, to R67 DHFR.NADPH appears weak as a titration in enthalpy is lost at low ionic strength. Steady-state kinetic studies for both wild type (wt) and K33M R67 DHFRs also support a strong electrostatic interaction between NADPH and the enzyme. Interestingly, quantitation of the observed salt effects by measuring the slopes of the log of ionic strength versus the log of k(cat)/K(m) plots indicates that only one ionic interaction is involved in forming the transition state. These data support a model where two ionic interactions are formed between NADPH and symmetry related K32 residues in the ground state. To reach the transition state, an ionic interaction between K32 and the pyrophosphate bridge is broken. This unusual scenario likely arises from the constraints imposed by the 222 symmetry of the enzyme.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

A flow microcalorimetric method for enzyme activity measurements: Application to dihydrofolate reductase

A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 × 10⁻⁴ units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.     

Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase

Isothermal titration calorimetry (ITC) was used to determine the thermodynamic driving force for inhibitor binding to the enzyme dihydrofolate reductase (DHFR) from Escherichia coli. 1,4-Bis-{[N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (1) binds DHFR:NADPH with a K(d) of 13±5 nM while the related inhibitor 1-{[N-(1-imino-guanidino-methyl)]sulfanylmethyl}-3-trifluoromethyl-benzene (2) binds DHFR:NADPH with a K(d) of 3.2±2.2 μM. The binding of these inhibitors has both a favorable entropy and enthalpy of binding. Additionally, we observe positive binding cooperativity between both 1 and 2 and the cofactor NADPH. Binding of compound 1 to DHFR is 285-fold tighter in the presence of the NADPH cofactor than in its absence. We did not detect binding of 2 to DHFR in the absence of NADPH. The backbone amide (1)H and (15)N NMR resonances of DHFR:NADPH and both DHFR:NADPH inhibitor complexes were assigned in order to better understand the binding of these inhibitors in solution. The chemical shift perturbations observed with the binding of 1 were greatest at residues closest to the binding site, but significant perturbations also occur away from the inhibitor location at amino acids in the vicinity of residue 58 and in the GH loop. The pattern of chemical shift changes observed with the binding of 2 is similar to that seen with 1. The main differences in chemical shift perturbation between the two inhibitors are in the Met20 loop and in residues at the interface between the inhibitor and NADPH.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.

The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.