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2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.

The state of protonation of substrates bound to mammalian dihydrofolate reductase (DHFR) has significance for the mechanism of catalysis. To investigate this, dihydrofolate and dihydropteroylpentaglutamate have been synthesized with {sup 15}N enrichment at N-5. {sup 15}N NMR studies have been performed on the binary complexes formed by bovine DHFR with these compounds and with (5-{sup 15}N)dihydrobiopterin. The results indicate that there is no protonation at N-5 in the binary complexes, and this was confirmed by {sup 13}C NMR studies with folate and dihydrofolate synthesized with {sup 13}C enrichment at C-6. The chemical shift displacements produced by complex formation are in the same direction as those which result from deprotonation of the N-3/C-4-O amide group and are consistent with at least partial loss of the proton from N-3. This would be possible if, as crystallographic data indicate, there is interaction of N-3 and the 2-amino group of the bound ligands with the carboxylate of the active site glutamate residue (Glu{sup 30}).

Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1)) of the ternary complex of the potent antitumor agent PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], reduced nicotinamide adenine dinucleotide phosphate (NADPH), and recombinant human dihydrofolate reductase (hDHFR) reveals multiple binding orientations for the hemiphthaloyl group of the inhibitor. Analysis of these data shows that PT523 binds with its pteridine ring in the same orientation observed for methotrexate (MTX) analogues. However, in each structure, the hemiphthaloyl ring occupies three alternate conformations. In the C2 lattice, the phthaloyl moiety binds in two extended conformations, A and C, with each conformer having a 180 degrees flip of the o-carboxylate group, and a third, lower occupancy conformer B, with the phthaloyl group folded within contact of the active-site pocket. In the orthorhombic lattice, PT523 also has three conformers for the phthaloyl group; however, these differ from those observed in the monoclinic lattice. Two major conformers, A and C, are displaced on either side of the extended position observed in the C2 lattice, one near the folded B conformer of the C2 lattice and the other extended. These conformers form tighter intermolecular contacts than those in the C2 lattice. Conformer B is folded back away from the active site in a unique position. There are also significant differences in the conformation of the adenine-ribose moiety of NADPH in both complexes that differ from that observed for other inhibitor-NADPH-hDHFR ternary complexes. These data suggest that the added intermolecular contacts made by the hemiphaloyl group of PT523 contribute to its tighter binding to hDHFR than MTX, which does not extend as far from the active site and cannot make these contacts. These crystallographic observations of multiple conformations for the hemiphthaloyl group are in general agreement with solution NMR data for the binding of PT523 to hDHFR [Johnson et al. (1997) Biochemistry 36, 4399-4411], which show that the hemiphthaloyl group may adopt more than one conformation. However, the crystallographic data reveal more discretely occupied positions than can be interpreted from the solution data. These results suggest that crystal packing interactions may influence their stability.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5-8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structures of dimeric and tetrameric R67 DHFR [Matthews et al. (1986) Biochemistry 25, 4194-4204] (Narayana, Matthews, and Xuong, personal communication), symmetry-related histidines 62, 162, 262, and 362 occur at the two dimer-dimer interfaces and protonation of these residues could destabilize tetrameric R67 DHFR. Ionization of these histidines was confirmed by monitoring the chemical shifts of the C2 proton in NMR experiments, and best fits of an incomplete titration curve yield a pKa of 6.77. Since tryptophans 38, 138, 238, and 338 also occur at the dimer-dimer interfaces, fluorescence additionally monitors the tetramer-two dimers equilibrium. When fluorescence was monitored over the pH range 5-8, a protein concentration dependence of fluorescence was observed and global fitting of three titration curves yielded Kd = 9.72 nM and pKa = 6.84 for the linked reactions: [formula: see text] Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200-600-fold decrease in catalytic efficiency. Decreased catalytic activity in dimeric R67 DHFR is presumably due to loss of the putative single active site pore found in tetrameric R67 DHFR.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.

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.

Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2{prime}-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K{sub m} values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K{sub m} and k{sub cat} values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K{sub m}(NADH)/K{sub m}(NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2{prime}-phosphate, makes a major contribution in terms of binding energy and differentiation of K{sub m} values for NADPH and NADH.more » Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme.« less

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.

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.

Dihydrofolate reductase from Streptococcus faecium has been labeled with (..gamma..-/sup 13/C)tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the /sup 13/C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup ..gamma../ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonancemore » 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity.« less

P3-(6-(N-Trifluoracetyl)aminohex-1-yl) deoxyguanosine triphosphate has been prepared by the reaction of N-trifluoroacetyl-6-aminohexanol 1-pyrophosphate with the imidazolide of dGMP and has been characterized. This compound and the corresponding free amine, obtained by removal of the protective trigluoroacetyl group, are activators of ribonucleotide reductase of Lactobacillus leichmannii. An affinity adsorbent for the reductase, prepared by reaction of the amine derivative with CNBr-activated Sepharose, contains dGTP covalently attached through the gamma-phosphate via a six-carbon chain to the matrix. The method of synthesis of the dGTP derivative is generally applicable to the synthesis of P3-(omega-aminoalk-1-yl)nucleoside triphosphate esters for the preparation of analogous affinity adsorbents. Ribonucleotide reductase can be rapidly purified to homogeneity, on a large scale, by use of dGTP-Sepharose and conditions for optimum recovery of the enzyme have been determined. The affinity of ribonucleotide reductase and other proteins for dGTP-Sepharose is increased by either raising the ionic strength or lowering the temperature of the eluent. Elution of the enzyme from the adsorbent can be achieved between pH 5.8 and 7.3, whereas at pH 5.3 the reductase is bound extremely tightly and cannot be recovered. Ribonucleotide reductase can be eluted from the adsorbent with dGTP or urea. Elution with urea is carried out at pH 6.3, where the enzyme is stable and maximum recovery is obtained. Affinity chromatography consistently produces ribonucleotide reductase of high specific activity (170-180 units/mg). In the presence of 0.1 to 1.2 M urea or hydroxyurea, the enzyme is inhibited, but allosteric activation is unchanged. No alteration in the structure or function of the reductase was detected when the enzyme was exposed to 2.0 M urea during elution from the affinity adsorbent, but exposure for longer periods causes some inactivation.

Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (pseudocoenzyme B12) purified from Clostridium tetanomorphum has been reacted with ribonucleotide reductase purified from Lactobacillus leichmannii under various conditions, and the properties of the products obtained have been compared by electron paramagnetic resonance (EPR) with those previously reported for products formed from the normal coenzyme (adenosylcobalamin). The rapidly formed intermediate and the slowly formed "doublet" species from the pseudocoenzyme have EPR spectra identical with those formed from the normal coenzyme. This and other considerations make it less likely that the unusual magnetic properties of the rapidly formed intermediate are due to strongly distorted octahedral symmetry about Co(II) as previously postulated. Instead it is probable that the EPR spectrum is due to interaction of the radical pair by both exchange coupling and magnetic dipole--dipole coupling. Although Coalpha-[alpha-(aden-9-YL)]cob(II)amide in solution does not show superhyperfine splitting in the EPR spectrum because of its base-off configuration, the cob(II)amide formed by degradation of the pseudocoenzyme within the catalytic site of the enzyme did show triplets due to a nitrogen axially coordinated to cobalt. This suggests that binding of the cob(II)amide to the reductase catalytic site causes a shift to the base-on form.

Dihydrofolate reductase isozyme 2 of Streptococcus faecium has been labeled with 13C in the C gamma position of tryptophan residues by growing the organism on a defined medium containing L-[gamma-13C]tryptophan (90% 13C). The 13C nuclear magnetic resonance (NMR) spectrum of the enzyme shows four well-resolved resonances which have nuclear Overhauser enhancements of 1.1-1.3. Values of T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) are significantly less than predicted for an isotropically rotating, rigid sphere with no intermolecular dipole-dipole interactions. Three of the resonances have chemical shifts downfield from the 13C resonance of urea-denatured enzyme by amounts up to 1.43 ppm. The chemical shift of resonance 4 in the spectrum is 4.0 ppm upfield from Trp C gamma of urea-denatured enzyme. This large upfield shift is attributed to electric field effects generated by polar side chains. The two more upfield peaks both provide evidence that the corresponding tryptophan residues, WC and WD, each undergo chemical exchange between alternative microenvironments. In the case of WC, which gives a resonance with two components, exchange is slow (ve, exchange rate much less than 55 s-1), and the relative populations of the two stable states are in the ratio 2:3. WD is apparently in intermediate to fast exchange on the NMR time scale. With a two-state model, ve increases from approximately 90 to 150 s-1 as the temperature is increased from 5 to 25 degrees C. This increases in temperature is also accompanied by an increase in the fractional population of the minor downfield state(s), from about 0.062 at 5 degrees C to 0.24 at 25 degrees C. However, the data may also be interpreted as a temperature-dependent equilibrium between a continuum of many states. WD is tentatively identified with Trp-22 since comparison of the sequences of Lactobacillus casei dihydrofolate reductase and S. faecium dihydrofolate reductase and inspection of the crystal structure of the L. casei enzyme indicate that Trp-6, Trp-115, and Trp-160 are probably all involved in regions of beta sheet whereas Trp-22 is in a loop joining beta A to alpha B. Earlier crystallographic evidence for the Escherichia coli reductase suggests that in the methotrexate complex with this enzyme the corresponding loop has a good deal of flexibility. It is probable that in the uncomplexed S. faecium reductase the motion of this loop is the major mechanism for the exchange process involving Trp-22. The upfield chemical shift of resonance 4 is attributed to electric field effects on C gamma of Trp-22 produced by the carboxylate groups of Asp-27 and Asp-9. On the basis of the small difference between the chemical shift of resonance 3 and that of tryptophan C gamma in urea-denatured reductase, it is suggested that WC may be identified with Trp-6.

Degradation of 7,8-dihydrofolate (H2folate) in the presence of dihydrofolate reductase (DHFR) has been shown due not to an oxygenase activity of the reductase as previously reported but to dismutation of H2folate to folate and 5,6,7,8-tetrahydrofolate (H4folate). The reaction can be followed spectrophotometrically or by analysis of the reaction mixture by high-performance liquid chromatography (HPLC). The products have also been isolated and characterized. Oxygen uptake during the reaction is much less than stoichiometric with H2folate disappearance and is attributed to autoxidation of the H4folate formed. The dismutation activity is a property of highly purified Streptococcus faecium DHFR isoenzyme 2 (but not isoenzyme 1) and of Lactobacillus casei DHFR, but not of bovine liver DHFR. The activity is dependent on tightly bound NADP+ and/or NADPH. Removal of the nucleotide results in loss of dismutation activity, which is restored by adding NADP+ or NADPH. Maximum activity is obtained when approximately 1 mol equiv of nucleotide is added per mol of DHFR. It is proposed that in the dismutation reaction bound NADP(H) is alternately reduced and oxidized by incoming molecules of H2folate with release of folate and H4folate, respectively. The relatively slow rate of folate formation presumably limits the rate of the overall reaction. The equilibrium constant for the dismutation reaction is 19.4 +/- 7.4 at 22 degrees C and pH 7.0. Calculation of standard oxidation-reduction potentials at pH 7 gave values of -0.230 V for the H2folate/H4 folate pair and -0.268 V for the folate/H2folate pair. The mechanism by which NADP+ is retained by the enzyme from some sources during purification procedures is unclear.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

(methyl-/sup 13/C)Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the /sup 13/C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T/sub 1/ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CH/sub 2/-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH/sub 2/-S bond is highly restricted so that the methyl group oscillates through a relatively narrow angular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably due to conformational transitionsmore » in the enzyme. However, NADP/sup +/ produced several changes,including movement of one resonance downfield by at least 1.7 ppM.« less

13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

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

The primary structure of dihydrofolate reductase from bovine liver has been established by Edman degradation of the intact carboxymethylated protein and of peptides obtained from the protein by the action of cyanogen bromide, trypsin, and the protease from Staphylococcus aureus, respectively. Since separation of some of the peptide mixtures by classical methods proved impossible, new systems were developed for the use of high-performance liquid chromatography to separate such mixtures. Some of the cleavage procedures used to obtain peptides gave atypical results at certain peptide bonds. The results are discussed in terms of the residues involved in these unexpectedly resistant or sensitive bonds. The sequence of the bovine liver enzyme is compared with those published for the enzyme from other sources, and known or probable functions of invariant residues are described. Sequences of vertebrate and bacterial reductases are compared and contrasted, and a possible role is considered for the residues which are invariant in bacterial reductases, but different in vertebrate reductases, in determining the selective inhibitory action of trimethoprim on bacterial reductases.