A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Kinetic investigation of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase

The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.     

Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase

Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.     

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.     

Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange.

The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis. The E. coli L28F mutant increased the pH-independent kcat from 11 to 50 s-1 but had little effect on Km(H2F). An increase in the rate constant for dissociation of H4F from E.H4F.NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat. Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1 with little perturbation of NADPH and NADP+ binding to E. Consequently, the flux efficiency of the E. coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain. The murine F31L mutant decreased the pH-independent kcat from 28 to 4.8 s-1 but had little effect on Km(H2F). A decrease in the rate constant for dissociation of H4F from E.H4F.NH (from 40 to 22 s-1) and E.H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in kcat. The rate constant for hydride transfer decreased from 9000 to 5000 s-1 with minor perturbation of NADPH binding. Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E. coli L28F mutant. This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.     

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.     

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.     

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.     

Impact on catalysis of secondary structural manipulation of the alpha C-helix of Escherichia coli dihydrofolate reductase

The alpha C-helix of Escherichia coli dihydrofolate reductase has been converted to its counterpart in Lactobacillus casei by a triple mutation in the helix (H45R, W47Y, and I50F). These changes result in a 2-fold increase in the steady-state reaction rate (kcat = 26 s-1) that is limited by an increased off rate for the release of tetrahydrofolate (koff = 40 s-1 versus 12 s-1). On the other hand the mutant protein exhibits a 10-fold increase in the KM value (6.8 microM) for dihydrofolate and a 10-fold decrease in the rate of hydride transfer (85 s-1) from NADPH to dihydrofolate. The elevated rate of tetrahydrofolate release upon the rebinding of NADPH, a characteristic of the wild-type enzyme-catalyzed reaction, is diminished. The intrinsic pKa (6.4) of the mutant enzyme binary complex with NADPH is similar to that of the wild type, but the pKa of the ternary complex is increased to 7.3, about on pH unit higher than the wild-type value. Further mutagenesis (G51P and an insertion of K52) was conducted to incorporate a hairpin turn unique to the C-terminus of the alpha C-helix of the L. casei enzyme in order to adjust a possible dislocation of the new helix. The resultant pentamutant enzyme shows restoration of many of the kinetic parameters, such as kcat (12 s-1), KM (1.1 microM for dihydrofolate), and khyd (526 s-1), to the wild-type values. The synergism in the product release is also largely restored. A substrate-induced conformational change responsible for the fine tuning of the catalytic process was found to be associated with the newly installed hairpin structure. The Asp27 residue of the mutant enzyme was found to be reprotonated before tetrahydrofolate release.     

Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues

Dihydrofolate reductase (DHFR) from a moderate thermophilic organism, Bacillus stearothermophilus, has been cloned and expressed. Physical characterization of the protein (BsDHFR) indicates that it is a monomeric protein with a molecular mass of 18,694.6 Da (0.8), coincident with the mass of 18 694.67 Da calculated from the primary sequence. Determination of the X-ray structure of BsDHFR provides the first structure for a monomeric DHFR from a thermophilic organism, indicating a high degree of conservation of structure in relation to all chromosomal DHFRs. Structurally based sequence alignment of DHFRs indicates the following levels of sequence identity and similarity for BsDHFR: 38 and 58% with Escherichia coli, 35 and 56% with Lactobacillus casei, and 23 and 40% with Thermotoga maritima, respectively. Steady state kinetic isotope effect studies indicate an ordered kinetic mechanism at elevated temperatures, with NADPH binding first to the enzyme. This converts to a more random mechanism at reduced temperatures, reflected in a greatly reduced K(m) for dihydrofolate at 20 degrees C in relation to that at 60 degrees C. A reduction in either temperature or pH reduces the degree to which the hydride transfer step is rate-determining for the second-order reaction of DHF with the enzyme-NADPH binary complex. Transient state kinetics have been used to study the temperature dependence of the isotope effect on hydride transfer at pH 9 between 10 and 50 degrees C. The data support rate-limiting hydride transfer with a moderate enthalpy of activation (E(a) = 5.5 kcal/mol) and a somewhat greater temperature dependence for the kinetic isotope effect than predicted from classical behavior [A(H)/A(D) = 0.57 (0.15)]. Comparison of kinetic parameters for BsDHFR to published data for DHFR from E. coli and T. maritima shows a decreasing trend in efficiency of hydride transfer with increasing thermophilicity of the protein. These results are discussed in the context of the capacity of each enzyme to optimize H-tunneling from donor (NADPH) to acceptor (DHF) substrates.     

Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.     

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)     

Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase.

R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6. Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism. Here, Raman difference measurements, conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive DHFR.NADPH.DHF complex, show that the pK(a) of N5 in the complex is less than 4. This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6. A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step. However, R67 shows that even without such a strategy an effective DHFR can still be designed.     

"Catch 222," the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69

R67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in Km values for both ligands and a 2-fold rise in the kcat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344-11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the Kd and Km values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type Km value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate.     

Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogues

Different classes of folate analogues have been examined with respect to the mechanism of their inhibition of dihydrofolate reductases from Escherichia coli and chicken liver. In addition, the degree of synergism between the binding of these compounds and NADPH has been investigated. Methotrexate acts as a slow, tight-binding inhibitor of both enzymes whereas trimethoprim is a slow, tight-binding inhibitor of the enzyme from E. coli and a classical inhibitor of the chicken-liver enzyme. Pyrimethamine, 2,4-diamino-6,7-dimethylpteridine, a phenyltriazine, folate and folinate exhibit classical inhibition. The degree of synergism between the binding of NADPH and the inhibitor varied from low for pyrimethamine and folate to very large for the phenyltriazine which binds to the chicken-liver enzyme almost 50 000-times more tightly in the presence of NADPH. The degree of synergism is reflected in the type of inhibition that the folate analogues yield with respect to NADPH. Compounds which exhibit slight synergism give noncompetitive inhibition whereas those with a high degree of synergism yield uncompetitive inhibition. With the exception of folinate, all compounds that act as classical inhibitors give rise to competitive inhibition with respect to dihydrofolate. Folinate exhibits competitive inhibition against NADPH and noncompetitive inhibition against dihydrofolate. These results are consistent with the formation of an enzyme-dihydrofolate-folinate complex. The (6S, alphaS)-diastereoisomer of folinate was bound at least 1000-times more tightly than the (6R, alphaS)-diastereoisomer. Consideration has been given to the possible interactions that occur between residues on the enzyme and groups on the inhibitor that give rise to slow-binding inhibition.     

Theoretical studies on the activation of the pterin cofactor in the catalytic mechanism of dihydrofolate reductase

Two mechanisms for facilitating hydride ion transfer from NADPH involving preprotonation of the pteridine rings of the dihydrofolate reductase substrates folate and dihydrofolate have been investigated by ab initio quantum mechanical methods. Protonation energies and effective solution pKas have been calculated for four protonated forms, three of which are nonpreferred in aqueous solution and therefore not directly accessible to experimental study. The pattern and degree of redistribution of the positive charge over the component rings of the N-heterobicyclic pi-system in these protonated forms have been analyzed in terms of changes in the electron populations of the ring atoms and total ring charges. The effects of such changes in promoting hydride ion transfer to C7 in folate and C6 in dihydrofolate have been evaluated by considering the extent of development of partial carbonium ion character at these carbon atoms and also the degree of electron deficiency in the pyrazine ring as a whole. The results illustrate that perturbations due, for instance, to protonation may be propagated by pi-electron coupling effects over medium-range distances of 4-6 A across the pteridine ring. The two mechanisms have been assessed in terms of the calculated absolute and relative pKas of the protonated species taking into account experimental information regarding possible stabilization of these forms in the enzyme active site and also the effectiveness of the various protonations in assisting the hydride ion transfer step. Judged against these criteria, the theoretical results favor the generally proposed mechanism involving preprotonation of N8 in folate and N5 in dihydrofolate. However, some support was also found for the alternative novel mechanism involving O4-protonation of both folate and dihydrofolate.     

Role of water in the catalytic cycle of E. coli dihydrofolate reductase

Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X-ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 A. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 A and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP+ and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.     

Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH

The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion of conformers are small (t1/2 for Ew----Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27 is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew----Et (t1/2 = 221 s) and an increased rate for Et----Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.