Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.     

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.     

The 1H-NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts

All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.     

Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate

Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques. Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure. Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues. Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis. The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments. As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex. Under these experimental conditions, only one bound form of the pterin ring could be detected.     

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

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.     

Comparative thermodynamic study of the interaction of some antifolates with dihydrofolate reductase

The thermodynamic parameters of the binding of antifolate drugs to bovine liver dihydrofolate reductase (EC 1.5.1.3., 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase) have been measured with a flow microcalorimetric method. These parameters are greatly influenced by the structure of the inhibitor and/or by the presence of NADPH and above all by temperature. For all the compounds studied, binding at 37 degrees C is driven by favourable enthalpy variations, whereas entropy variations are unfavourable. At 10 degrees C, reactions are both enthalpically and entropically driven. These effects can be explained by a partial thermal denaturation of dihydrofolate reductase at 37 degrees C, which is restructured by NADPH and/or the antifolate. The refolding induced by the antifolate trimetrexate may explain its high association constant in the binary system (without NADPH), and the weaker cooperative effect of NADPH in the ternary system, as compared to methotrexate. In contrast, the poor affinity of trimethoprim for mammalian dihydrofolate reductase in binary and ternary systems at 37 degrees C is the result of a weaker stabilizing effect of this compound as regards temperature increase. Heat capacity variation linked to the complex formation reaction showed that this conformational transition is more pronounced between 25 and 37 degrees C than between 10 and 25 degrees C. Thus, the ability of the inhibitors to give to dihydrofolate reductase a more stable thermal behaviour at 37 degrees C is determinant in their binding.     

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli

Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers. However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale. Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

Selenomethionyl dihydrofolate reductase from Escherichia coli. Comparative biochemistry and 77Se nuclear magnetic resonance spectroscopy.

The biosynthetic replacement of Met residues by selenomethionine (SeMet) facilitates the determination of three-dimensional structure by multiwavelength anomalous diffraction (Yang, W., Hendrickson, W. A., Crouch, R.J., and Satow, Y. (1990) Science 249, 1398-1405). In an effort to examine any biochemical effects due to the replacement of Met residues by SeMet, we chose to compare the kinetic and binding properties of selenomethionyl dihydrofolate reductase with those of the wt enzyme. There are 5 Met residues in Escherichia coli dihydrofolate reductase with 2 located in the Met-20 loop, which is a sequence of residues forming a lid over the active site. Utilizing plasmid pWT8, which affords 10-15% soluble protein as E. coli dihydrofolate reductase, we readily isolated both the SeMet and wt enzymes from E. coli DL41 utilizing a novel purification protocol. Both enzymes exhibited essentially the same kinetic and binding properties, including specific activities (45 mumol/min/mg), Km (7,8-dihydrofolate = 0.39 microM; NADPH = 2.0 microM), kcat (13.5/s), and 1:1 noncovalent inhibitory binding ratios with methotrexate. The inhibitory effects of divalent and monovalent cations on activity were also assessed, with the SeMet-containing enzyme exhibiting a uniformly greater sensitivity than the wt enzyme. We conclude that the biochemical properties of dihydrofolate reductase are virtually unperturbed by SeMet inclusion. Analysis of SeMet dihydrofolate reductase by 77Se nuclear magnetic resonance spectroscopy revealed five distinct resonances, thus indicating the potential value of this technique in employing selenium as a nonperturbing NMR probe of protein structure and function.     

A sensitive high-performance liquid chromatographic-fluoromotric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to ≥ 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 μg). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 n methotrexate. The K_m of the enzyme for NADPH was found to be 7.5 μ , while the K_m for 7,8-dihydrobiopterin was 88 μ . Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis

In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors. Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant. Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase. The serine 31 mutation had little effect on the steady-state kinetic properties of the enzyme but produced a 100-fold increase in the dissociation constant (Kd) for the inhibitor methotrexate. The serine 34 mutant had much greater alterations in its properties than S31; specifically, S34 had a 3-fold reduction in the Km for NADPH, a 24-fold increase in the Km for H2folate, a 3-fold reduction in the overall reaction rate kcat, and an 80,000-fold increase in the Kd for methotrexate. In addition, the pH dependence of the steady-state kinetic parameters of S34 were different from that of the wild-type enzyme. These results suggest that phenylalanine 31 and phenylalanine 34 make very different contributions to ligand binding and catalysis in the human dihydrofolate reductase.     

Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase

Dihydrofolate reductase has been purified from a trimethoprim-resistant strain of Neisseria gonorrhoeae. The enzyme showed a single component on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 18,000) and on isoelectric focusing in 5 M urea (pI = 6.8). Although gel electrophoresis under nondenaturing conditions resolved the preparation into two enzymatically active proteins (called form 1 and form 2), they were not genetically determined isozymes. Both had a similar dihydrofolate Km (2 microM), NADPH Km (10 microM), and trimethoprim Ki (20 nM), and form 2 (the slower migrating species) was shown to be generated from form 1 by the electrophoresis conditions. The complete covalent structure of the enzyme has also been determined. It is a single polypeptide composed of 162 residues and containing 4 cysteines. The gonococcal dihydrofolate reductase shares a 35% homology with the chicken liver enzyme and a 40% homology with the Escherichia coli enzyme. Most of these identities are residues that have been implicated in the binding of NADPH and methotrexate to the E. coli and Lactobacillus casei reductases.     

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)