Thermodynamics and solvent effects on substrate and cofactor binding in Escherichia coli chromosomal dihydrofolate reductase

Chromosomal dihydrofolate reductase from Escherichia coli catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. The thermodynamics of ligand binding were examined using an isothermal titration calorimetry approach. Using buffers with different heats of ionization, zero to a small, fractional proton release was observed for dihydrofolate binding, while a proton was released upon NADP(+) binding. The role of water in binding was additionally monitored using a number of different osmolytes. Binding of NADP(+) is accompanied by the net release of ～5-24 water molecules, with a dependence on the identity of the osmolyte. In contrast, binding of dihydrofolate is weakened in the presence of osmolytes, consistent with "water uptake". Different effects are observed depending on the identity of the osmolyte. The net uptake of water upon dihydrofolate binding was previously observed in the nonhomologous R67-encoded dihydrofolate reductase (dfrB or type II enzyme) [Chopra, S., et al. (2008) J. Biol. Chem. 283, 4690-4698]. As R67 dihydrofolate reductase possesses a nonhomologous sequence and forms a tetrameric structure with a single active site pore, the observation of weaker DHF binding in the presence of osmolytes in both enzymes implicates cosolvent effects on free dihydrofolate. Consistent with this analysis, stopped flow experiments find betaine mostly affects DHF binding via changes in k(on), while betaine mostly affects NADPH binding via changes in k(off). Finally, nonadditive enthalpy terms when binary and ternary cofactor binding events are compared suggest the presence of long-lived conformational transitions that are not included in a simple thermodynamic cycle.     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Site-specific mutagenesis of dihydrofolate reductase from Escherichia coli

Two site-specific mutations of dihydrofolate reductase from Escherichia coli based on the x-ray crystallographic structure were constructed. The first mutation (His-45----Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr-113----Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5-C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6-40-fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild-type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20-30-fold, and presumably dihydrofolate also, with a corresponding 20-30-fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.     

The electrostatic potential of Escherichia coli dihydrofolate reductase

Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of -10 electrons yet it binds ligands with net charges of -4 (NADPH) and -2 (folate or dihydrofolate). Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished. The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside "pockets" of positive potential that enable the enzyme to bind the negatively charged ligands. The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme. The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent-protein interface, is analogous to the situation occurring in superoxide dismutase. Thus DHFR is another case where the shape of the active site focuses electric fields out into solution. The positive electrostatic potential at the entrance of the ligand binding site in E. coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57--residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate. The latter has been postulated to be involved in the catalytic process. A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species. It is noted that, although the net charges of DHFRs from different species vary from +3 to -10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli

The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L.     

Characterization of the cloned Escherichia coli dihydrofolate reductase

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.     

Purification and properties of Escherichia coli dihydrofolate reductase.

Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.     

Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase

A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C. Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH. The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step. The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate. The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex.     

Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase

Full time course studies of the kinetic activity of Escherichia coli dihydrofolate reductase show that there is an increase in activity with time. The half-time for this hysteretic behavior is about 9 s. Preincubation of the enzyme with either of the substrates abolishes the lag and results in initial velocities which are 2-2.3-fold faster than those observed for the non-preincubated enzyme. The kinetic properties of the activated and nonactivated forms of the enzyme appear to be similar as measured by the full time course of the reaction. The results are consistent with observations for NADPH binding studies that the enzyme exists in two interconvertible forms, one of which is incapable of binding NADPH (Cayley, P. J., Dunn, S. M. J., and King, R. W. (1981) Biochemistry 20, 874-879).     

Hydrophobic interactions via mutants of Escherichia coli dihydrofolate reductase: separation of binding and catalysis

The strictly conserved residue leucine-54 of Escherichia coli dihydrofolate reductase forms part of the hydrophobic wall which binds the p-aminobenzoyl side chain of dihydrofolate. In addition to the previously reported glycine-54 mutant, isoleucine-54 and asparagine-54 substitutions have been constructed and characterized with regard to their effects on binding and catalysis. NADP+ and NADPH binding is virtually unaffected with the exception of a 15-fold decrease in NADPH dissociation from the Gly-54 mutant. The synergistic effect of NADPH on tetrahydrofolate dissociation seen in the wild-type enzyme is lost in the isoleucine-54 mutant: little acceleration is seen in tetrahydrofolate dissociation when cofactor is bound, and there is no discrimination between reduced and oxidized cofactor. The dissociation constants for dihydrofolate and methotrexate increase in the order Leu less than Ile less than Asn less than Gly, varying by a maximum factor of 1700 for dihydrofolate and 6300 for methotrexate. Despite these large changes in binding affinity, the hydride transfer rate of 950 s-1 in the wild-type enzyme is decreased by a constant factor of ca. 30 (2 kcal/mol) regardless of the mutant. Thus, the contributions of residue 54 to binding and catalysis appear to have been separated.     

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.