The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases

A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

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)     

Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.

The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E. coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.     

Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium.

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.     

E. coli MEP synthase: steady-state kinetic analysis and substrate binding.

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.     

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

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

A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Gammaherpesviruses encode functional dihydrofolate reductase activity

We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV). All three enzymes proved catalytically active. The K(m) value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02+/-0.44 microM, that of HVS DHFR was 4.31+/-0.56 microM, and that of RRV DHFR is 7.09+/-0.11 microM. These values are approximately 5-15-fold higher than the K(m) value reported for the human DHFR. The K(m) value of HHV-8 DHFR for NADPH was 1.31+/-0.23 microM, that of HVS DHFR was 3.78+/-0.61 microM, and that of RRV DHFR was 7.47+/-0.59 microM. These values are similar or slightly higher than the corresponding K(m) value of the human enzyme. Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gammaherpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme.     

Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

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)     

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.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K(d) values) for NADPH (0.87 microM), NADP(+) (16 microM), NADH (50 microM), and NAD(+) (100-500 microM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K(d) values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K(d) values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidized.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.     

Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.     

Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.