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.     

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 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.     

R67, the other dihydrofolate reductase: rational design of an alternate active site configuration

R67 dihydrofolate reductase (DHFR) bears no sequence or structural homologies with chromosomal DHFRs. The gene for this enzyme produces subunits that are 78 amino acids long, which assemble into a homotetramer possessing 222 symmetry. More recently, a tandem array of four gene copies linked in-frame was constructed, which produces a monomer containing 312 amino acids named Quad3. Asymmetric mutations in Quad3 have also been constructed to probe the role of Q67 and K32 residues in catalysis. This present study mixes and matches mutations to determine if the Q67H mutation, which tightens binding approximately 100-fold to both dihydrofolate (DHF) and NADPH, can help rescue the K32M mutation. While the latter mutation weakens DHF binding over 60-fold, it concurrently increases kcat by a factor of 5. Two Q67H mutations were added to gene copies 1 and 4 in conjunction with the K32M mutation in gene copies 1 and 3. Addition of these Q67H mutations tightens binding 40-fold, and the catalytic efficiency (kcat/Km(DHF)) of the resulting protein is similar to that of Quad3. Since these Q67H mutations can mostly compensate for the K32M lesion, K32 must not be necessary for DHF binding. Another multimutant combines the K32M mutation in gene copies 1 and 3 with the Q67H mutation in all gene copies. This mutant is inhibited by DHF but not NADPH, indicating that NADPH binds only to the wild type half of the pore, while DHF can bind to either the wild type or mutant half of the pore. This inhibition pattern contrasts with the mutant containing only the Q67H substitution in all four gene copies, which is severely inhibited by both NADPH and substrate. Since gene duplication and divergence are evolutionary tools for gaining function, these constructs are a first step toward building preferences for NADPH and DHF in each half of the active site pore of this primitive enzyme.     

Role of ionic interactions in ligand binding and catalysis of R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR), which catalyzes the NADPH dependent reduction of dihydrofolate to tetrahydrofolate, belongs to a type II family of R-plasmid encoded DHFRs that confer resistance to the antibacterial drug trimethoprim. Crystal structure data reveals this enzyme is a homotetramer that possesses a single active site pore. Only two charged residues in each monomer are located near the pore, K32 and K33. Site-directed mutants were constructed to probe the role of these residues in ligand binding and/or catalysis. As a result of the 222 symmetry of this enzyme, mutagenesis of one residue results in modification at four related sites. All mutants at K32 affected the quaternary structure, producing an inactive dimer. The K33M mutant shows only a 2-4-fold effect on K(m) values. Salt effects on ligand binding and catalysis for K33M and wildtype R67 DHFRs were investigated to determine if these lysines are involved in forming ionic interactions with the negatively charged substrates, dihydrofolate (overall charge of -2) and NADPH (overall charge of -3). Binding studies indicate that two ionic interactions occur between NADPH and R67 DHFR. In contrast, the binding of folate, a poor substrate, to R67 DHFR.NADPH appears weak as a titration in enthalpy is lost at low ionic strength. Steady-state kinetic studies for both wild type (wt) and K33M R67 DHFRs also support a strong electrostatic interaction between NADPH and the enzyme. Interestingly, quantitation of the observed salt effects by measuring the slopes of the log of ionic strength versus the log of k(cat)/K(m) plots indicates that only one ionic interaction is involved in forming the transition state. These data support a model where two ionic interactions are formed between NADPH and symmetry related K32 residues in the ground state. To reach the transition state, an ionic interaction between K32 and the pyrophosphate bridge is broken. This unusual scenario likely arises from the constraints imposed by the 222 symmetry of the enzyme.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

A balancing act between net uptake of water during dihydrofolate binding and net release of water upon NADPH binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH.DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.     

Breaking symmetry: mutations engineered into R67 dihydrofolate reductase,a D2 symmetric homotetramer possessing a single active site pore

R67 dihydrofolate reductase (DHFR) is an enzyme, encoded by an R-plasmid, that confers resistance to the antibacterial agent trimethoprim. This homotetramer possesses a single active site pore and exact 222 symmetry. The symmetry imposes constraints on the ability of the enzyme to optimize binding of the substrate, dihydrofolate (DHF), and the cofactor, NADPH, resulting in a "one site fits both ligands" approach. This approach allows formation of either a NADPH.NADPH, dihydrofolate.dihydrofolate, or NADPH.dihydrofolate complex. The first two complexes are nonproductive, while the third is the productive catalytic species. To break the symmetry of the active site, a tandem array of four R67 DHFR genes has been linked in frame, allowing individual manipulation of each gene copy. Various numbers and combinations of asymmetric Q67H mutations have been engineered into the tandem gene array. The Q67H mutation was chosen for investigation as it was previously found to tighten binding to both dihydrofolate and NADPH by approximately 100-fold in homotetrameric R67 DHFR [Park, H., Bradrick, T. D., and Howell, E. E. (1997) Protein Eng. 10, 1415-1424]. Nonadditive effects on ligand binding are observed when one to four mutations are inserted, indicating either conformational changes in the protein or different cooperativity patterns in the ligand-ligand interactions. From steady state kinetics, addition of Q67H mutations does not drastically affect formation of the NADPH.dihydrofolate complex; however, a large energy difference between the productive and nonproductive complexes is no longer maintained. A role for Q67 in discriminating between these various states is proposed. Since theories of protein evolution suggest gene duplication followed by accumulation of mutations can lead to divergence of activity, this study is a first step toward asking if introduction of asymmetric mutations in the quadrupled R67 DHFR gene can lead to optimization of ligand binding sites.     

"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.     

Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.     

Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization.

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4). Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

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.     

Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on L-arginine hydroxylation and GN-hydroxy-L-arginine oxidation

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of L-arginine (Arg) with formation of an intermediate, GN-hydroxy-L-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H(4)B and instability of the heme-CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H(4)B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 microM NHA and 0.39 microM H(4)B to reach the half-maximal NHA-driven NO formation rate (EC(50)), while R365L needed 59 microM NHA and 0.73 microM H(4)B to achieve EC(50). The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.     

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

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

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.     

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.     

Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.