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.     

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.     

Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pKa of the bound dihydrofolate

We evaluate the pK(a) of dihydrofolate (H(2)F) at the N(5) position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP(+):H(2)F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H(2)F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pK(a) is modulated by the Met20 loop fluctuations, providing the largest pK(a) shift in substates with a "tightly closed" loop conformation; in the "partially closed/open" substates, the pK(a) is similar to that in the occluded complex. Conducive to the protonation, tightly closing the Met20 loop enhances the interactions of the cofactor and the substrate with the Met20 side chain and aligns the nicotinamide ring of the cofactor coplanar with the pterin ring of the substrate. Overall, the present study favors the hypothesis that N(5) is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Envisioning the Loop Movements and Rotation of the Two Subdomains of Dihydrofolate Reductase by Elastic Normal Mode Analysis

Abstract

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.     

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.     

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

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

Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.     

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

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

Crystal structures of shikimate dehydrogenase AroE from Thermus thermophilus HB8 and its cofactor and substrate complexes: insights into the enzymatic mechanism

Shikimate dehydrogenase (EC 1.1.1.25) catalyses the fourth step of the shikimate pathway which is required for the synthesis of the aromatic amino acids and other aromatic compounds in bacteria, microbial eukaryotes, and plants. The crystal structures of the shikimate dehydrogenase AroE from Thermus thermophilus HB8 in its ligand-free form, binary complexes with cofactor NADP+ or substrate shikimate, and the ternary complex with both NADP(H) and shikimate were determined by X-ray diffraction method at atomic resolutions. The crystals are nearly isomorphous with the asymmetric unit containing a dimer, each subunit of which has a bi-domain structure of compact alpha/beta sandwich folds. The two subunits of the enzyme display asymmetry in the crystals due to different relative orientations between the N- and C-terminal domains resulting in a slightly different closure of the interdomain clefts. NADP(H) is bound to the more closed form only. This closed conformation with apparent higher affinity to the cofactor is also observed in the unliganded crystal form, indicating that the NADP(H) binding to TtAroE may follow the selection mode where the cofactor binds to the subunit that happens to be in the closed conformation in solution. Crystal structures of the closed subunits with and without NADP(H) show no significant structural difference, suggesting that the cofactor binding to the closed subunit corresponds to the lock-and-key model in TtAroE. On the other hand, shikimate binds to both open and closed subunit conformers of both apo and NADP(H)-liganded holo enzyme forms. The ternary complex TtAroE:NADP(H):shikimate allows unambiguous visualization of the SDH permitting elucidation of the roles of conserved residues Lys64 and Asp100 in the hydride ion transfer between NADP(H) and shikimate.     

The crystal structure of Escherichia coli ketopantoate reductase with NADP+ bound

The NADPH-dependent reduction of ketopantoate to pantoate, catalyzed by ketopantoate reductase (KPR; EC 1.1.1.169), is essential for the biosynthesis of pantothenate (vitamin B(5)). Here we present the crystal structure of Escherichia coli KPR with NADP(+) bound, solved to 2.1 A resolution. The cofactor is bound in the active site cleft between the N-terminal Rossmann-fold domain and the C-terminal alpha-helical domain. The thermodynamics of cofactor and substrate binding were characterized by isothermal titration calorimetry. The dissociation constant for NADP(+) was found to be 6.5 muM, 20-fold larger than that for NADPH (0.34 muM). The difference is primarily due to the entropic term, suggesting favorable hydrophobic interactions of the more lipophilic nicotinamide ring in NADPH. Comparison of this binary complex structure with the previously studied apoenzyme reveals no evidence for large domain movements on cofactor binding. This observation is further supported both by molecular dynamics and by calorimetric analysis. A model of the ternary complex, based on the structure presented here, provides novel insights into the molecular mechanism of enzyme catalysis. We propose a conformational switch of the essential Lys176 from the "resting" state observed in our structure to an "active" state, to bind ketopantoate. Additionally, we identify the importance of Asn98 for substrate binding and enzyme catalysis.     

Crystal structure of a type II dihydrofolate reductase catalytic ternary complex

Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR.DHF.NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.     

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

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

Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on (15)N and (2)H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in μs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect.     

Interloop contacts modulate ligand cycling during catalysis by Escherichia coli dihydrofolate reductase

As a continuation to our studies on the importance of interloop interactions in the Escherichia coli DHFR catalytic cycle, we have investigated the role of the betaG-betaH loop in modulating the closed and occluded conformations of the Met20 loop during the DHFR catalytic cycle. Specifically, to assess the importance of the hydrogen bond formed between Ser148 in the betaG-betaH loop and the Met20 loop, Ser148 was independently substituted with aspartic acid, alanine, and lysine. Moreover, the betaG-betaH loop was deleted entirely to yield the Delta(146-148) DHFR mutant. Steady-state turnover rates for all mutants were at most 3-fold lower than the wild-type rate. Lack of an isotope effect on this rate indicated the chemistry step does not contribute to the steady-state turnover. Consistent with this finding, hydride transfer rates for the DHFR mutants were at least 10-fold greater than the observed steady-state rates. The values ranged from a 30% decrease (Ser148Ala and Ser148Lys) to a 50% increase (Ser148Asp) in rate relative to that of the wild type. Modifications of the betaG-betaH loop enhanced the affinity for the cofactor and decreased the affinity for pterin, as determined by the K(D) values of the mutant proteins. Further analysis of Ser148Ala and Delta(146-148) DHFRs indicated these effects were manifest mainly in ligand off rates, although in some cases the on rate was affected. The Ser148Asp and Delta(146-148) mutations perturbed the preferred catalytic cycle through the introduction of branching at key intermediates. Rather than following the single WT pathway which involves loss of NADP(+) and rebinding of NADPH to precede loss of the product H4F (negative cooperativity), the mutants can reenter the catalytic cycle through different pathways. These findings suggest that the role of the interloop interaction between the betaG-betaH loop and the Met20 loop is to modulate ligand off rates allowing for proper cycling through the preferred kinetic pathway.     

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.     

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.     

A review of the binding-change mechanism for proton-translocating transhydrogenase

Proton-translocating transhydrogenase is found in the inner membranes of animal mitochondria, and in the cytoplasmic membranes of many bacteria. It catalyses hydride transfer from NADH to NADP(+) coupled to inward proton translocation. Evidence is reviewed suggesting the enzyme operates by a "binding-change" mechanism. Experiments with Escherichia coli transhydrogenase indicate the enzyme is driven between "open" and "occluded" states by protonation and deprotonation reactions associated with proton translocation. In the open states NADP(+)/NADPH can rapidly associate with, or dissociate from, the enzyme, and hydride transfer is prevented. In the occluded states bound NADP(+)/NADPH cannot dissociate, and hydride transfer is allowed. Crystal structures of a complex of the nucleotide-binding components of Rhodospirillum rubrum transhydrogenase show how hydride transfer is enabled and disabled at appropriate steps in catalysis, and how release of NADP(+)/NADPH is restricted in the occluded state. Thermodynamic and kinetic studies indicate that the equilibrium constant for hydride transfer on the enzyme is elevated as a consequence of the tight binding of NADPH relative to NADP(+). The protonation site in the translocation pathway must face the outside if NADP(+) is bound, the inside if NADPH is bound. Chemical shift changes detected by NMR may show where alterations in protein conformation resulting from NADP(+) reduction are initiated. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.     

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.