Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.     

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618-12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.     

Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent

Mutations in Dihydrofolate Reductase (dhfr) gene of Plasmodium vivax are known to be associated with resistance to antifolate drugs. To analyze the extent of these mutations in P. vivax population in India, dhfr gene was isolated and sequenced for 121 P. vivax isolates originating from different geographical regions of Indian subcontinent. These sequences were compared with the gene sequence that represent wild type sequence (accession no. X98123). P. vivax dhfr (Pvdhfr) sequences showed limited polymorphism and about 70% isolates showed wild type dhfr sequence. A total of 36 mutations were found at 11 positions in 121 isolates. A majority of mutant isolates showed double mutations at residues 58 (S-->R) and 117 (S-->N), known to be associated with pyrimethamine resistance, but only 19% showed double mutations at residues 57 (F-->L) and 58 (S-->R). Pvdhfr alleles showing quadruple mutation (F57L, S58R, T61M and S117T) were found in two isolates. Three other mutations reported earlier at residue 13, 33 and 173 were not found in any of the Isolates. Six novel mutations at residues 38 (R-->G), 93 (S-->C), 109 (S-->H), 131 (R-->G), 159 (V-->A) and 188 (I-->V) were observed in seven isolates. Whether these novel mutations are linked to pyrimethamine resistance remains to be established.     

Differential effect of regional drug pressure on dihydrofolate reductase and dihydropteroate synthetase mutations in southern Mozambique

The prevalence and frequency of the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) mutations associated with sulfadoxine-pyrimethamine (SP) resistance at 13 sentinel surveillance sites in southern Mozambique were examined regularly between 1999 and 2004. Frequency of the dhfr triple mutation increased from 0.26 in 1999 to 0.96 in 2003, remaining high in 2004. The dhps double mutation frequency peaked in 2001 (0.22) but declined to baseline levels (0.07) by 2004. Similarly, parasites with both dhfr triple and dhps double mutations had increased in 2001 (0.18) but decreased by 2004 (0.05). The peaking of SP resistance markers in 2001 coincided with a SP-resistant malaria epidemic in neighboring KwaZulu-Natal, South Africa. The decline in dhps (but not dhfr) mutations corresponded with replacement of SP with artemether-lumefantrine as malaria treatment policy in KwaZulu-Natal. Our results show that drug pressure can exert its influence at a regional level rather than merely at a national level.     

Molecular surveillance of mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum in Ethiopia

Point mutations in the genes for dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) of Plasmodium falciparum isolates are associated with sulfadoxine/pyrimethamine (SP) treatment failure, respectively. This study was conducted to assess the prevalence of SP resistance in P. falciparum isolates collected at the Jimma Health Center in southwestern Ethiopia. In this study, the genetic profile of P. falciparum isolates with respect to DHFR and DHPS genes was assessed in 124 individuals. The prevalence of single, double, and multiple mutations in these genes was calculated. The sequence profile showed that all samples carried a double mutation at the positions 51 and 108 (I51N108) in the DHFR gene. Sixty-seven (54.03%) of the isolates had an additional third mutation at position 59, resulting in the triple mutant I51R59N108. All isolates carried mutations G437 and E540 in the DHPS gene. Two isolates (1.61%) had additional mutations at codon 581 (A581).     

Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area.     

Detection of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) gene by dot-blot hybridization

There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.     

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.     

Pyrimethamine-sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali.

To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 190 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days. Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect DHFR and DHPS mutations on filter paper blood samples from pretreatment and post-treatment infections. Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed. Post-PS treatment infections had significantly higher rates of DHFR mutations at codons 108 and 59. No significant selection for DHPS mutations was seen. Pyrimethamine-sulfadoxine is highly efficacious in Mali, and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance, rapid selection of DHFR mutations supports their role in PS failure.     

Lipid-soluble diaminopyrimidine inhibitors of dihydrofolate reductase.

On the basis of activity against experimental tumors and potency as inhibitors of human dihydrofolate reductase, two compounds were selected for pharmacokinetic evaluation: metoprine ((2,4-diamino-5-(3',4'-dichlorophenyl)-6-methyl pyrimidine, DDMP, B.W. 197U) and etoprine, the corresponding 6-ethyl analog (DDEP, B.W. 276U). These lipid-soluble compounds readily cross the blood-brain barrier and penetrate rapidly into brain and brain tumors induced in rats by ethylnitrosourea. Both compounds are extensively bound to human plasma protein and their slow elimination from plasma and tissues contrasts with the kinetics of methotrexate. Cerebrospinal fluid levels of "folate" were elevated following oral administration of citrovorum factor to rats but not following equivalent doses of folic acid. The balance between selective action of the drug and selective protection by the vitamin is discussed with regard to differential distribution into separate compartments.     

An essential intermediate in the folding of dihydrofolate reductase

The folding of Escherichia coli dihydrofolate reductase was examined at pH 7.8 and 15 degrees C by using stopped-flow fluorescence and absorbance spectroscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow absorbance spectroscopy using methotrexate binding assays shows a distinct lag phase during these time frames for the native state. The lag in absorbance kinetics and the lack of fast-track folding events indicate that the formation of this ensemble of intermediates is an obligatory step in the folding reaction.     

Conformational effects of environmentally induced, cancer-related mutations in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. A considerable number of environmentally induced, cancer-related p53 mutations in human tumors have been found in a highly conserved proline-rich sequence of the p53 protein encompassed by amino acid residues 147-158. Using conformational energy analysis based on ECEPP (Empirical Conformational Energy for Peptides Program), we have determined the low-energy three-dimensional structures for this dodecapeptide sequence for the human wild-type p53 protein and three environmentally induced, cancer-related mutant p53 proteins with His-151, Ser-152, and Val-154, respectively. The results suggest that the wild-type sequence adopts a well-defined low-energy conformation and that the mutant peptides adopt well-defined conformations that are distinctly different from the conformation of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of these environmentally induced, cancer-related, mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

Point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum and resistance to sulfadoxine-pyrimethamine in Sri Lanka

Sulfadoxine-pyrimethamine (SP) is the second-line treatment for Plasmodium falciparum malaria in Sri Lanka. Resistance to SP is caused by point mutations in the dihydrofolate reductase (Pf-dhfr) and dihydropteroate synthase (Pf-dhps) genes of P. falciparum. We determined the genotype of Pf-dhfr and Pf-dhps and the clinical response to SP in 30 field isolates of P. falciparum from Sri Lanka. All patients treated with SP had an adequate clinical response. Eighty-five percent (23 of 27) of pure field isolates carried parasites with double mutant alleles of Pf-dhfr (C59R + S108N) and showed about 200-fold higher levels of resistance to pyrimethamine than the wild type in a yeast system. None of the isolates had either known or novel mutations at other positions in the dhfr domain. In contrast, 67% (20 of 30) of the isolates carried parasites that were wild type for Pf-dhps. In Sri Lanka, detection of the triple mutant allele of Pf-dhfr will require tracking mutations at codon 51.     

Dynamics of trimethoprim bound to dihydrofolate reductase.

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.     

Short communication: point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Colombia

Point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum can lead to an increasing resistance of P. falciparum to pyrimethamine/sulphadoxine. We examined the prevalence of these mutations in 36 samples from Colombia. Analysed by polymerase chain reaction (PCR) for infection with P. falciparum, 25 (69%) tested positive. These positive isolates were tested further for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and following mutation-specific restriction enzyme digestion. Gene mutations occurred in both the DHFR and DHPS gene of the Colombian isolates, suggesting that resistance to antifolate drugs exists or may develop soon in Colombia.     

Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use

Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations (P<.001). In addition to suggesting that there is less selective pressure on DHFR than on DHPS, this study reinforces the hypothesis that mutations in the DHPS gene are likely involved in the development of sulfa resistance in P. carinii.     

Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate

The interaction of dihydrofolate (H(2)F) and NADPH with a fluorescent derivative of H(2)F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H(2)F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 x 10(7) M(-1).s(-1), and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H(2)F with a bimolecular rate constant of 2.6-5.7 x 10(6) M(-1).s(-1), with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s(-1) at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme-substrate complexes exist.     

Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.     

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy (¹⁵N, ¹³C, ²H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for “promoting motions” in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate.There is heated debate about the role of protein dynamics in enzyme catalysis, especially for reactions that involve transfer of hydrogen (H⁺, H·, H^–), in which quantum tunneling is significant. It has been suggested that “promoting protein motions”, i.e., specific fluctuations that might reduce the barrier height or promote tunneling by reducing donor–acceptor distances, can drive enzymatic reactions (1, 2). Such models include promoting vibrations (3), environmentally coupled tunneling (1), and vibrationally enhanced ground-state tunneling (4). Several of these proposals suggest that the anomalous temperature and pressure dependences of experimentally observed reaction rates and kinetic isotope effects are the consequence of protein motions on the pico- to femtosecond timescale that reduce the width and/or height of the potential energy barrier along the chemical reaction coordinate. However, a connection between promoting motions and potential energy barrier modulation has never been demonstrated directly, and recent work has shown that the temperature dependence of kinetic isotope effects can be accounted for by conformational effects for a number of enzymes (5). Whereas some authors postulate dynamics as a key driving force in catalysis (1–4), others have performed analyses showing activation free-energy reduction, which is an equilibrium property, to be the source of catalysis (6–14). Enzyme reactions, and particularly their dynamics, present formidable challenges for study, and progress requires a combination of theoretical, experimental, and computational approaches (5, 15–18).Dihydrofolate reductase (DHFR) has been at the heart of the debates about the relationship between enzyme dynamics and catalysis. DHFR catalyses the NAPDH-dependent reduction of 7,8-dihydrofolate (H₂F) to 5,6,7,8-tetrahydrofolate (H₄F) by hydride transfer from C4 of NADPH and protonation of N5 of H₂F. The enzyme from Escherichia coli (EcDHFR) cycles through five reaction intermediates, namely E·NADPH, E·NADPH·H₂F, E·NADP⁺·H₄F, E·H₄F, and E·NADPH·H₄F (19), and adopts two major conformations, the closed conformation in the reactant complexes E·NADPH and E·NADPH·H₂F and the occluded conformation in the three product complexes E·NADP⁺·H₄F, E·H₄F, and E·NADPH·H₄F (20). The physical steps of ligand association and dissociation have been shown to depend on movements between these two conformations (20, 21). The actual chemical step of hydride transfer from NADPH to H₂F occurs with a reaction-ready configuration of the closed complex (Fig. 1), where the M20 loop (residues 8–23) closes over the active site to shield the reactants from solvent and provide an optimal geometry and electrostatic environment of the active site for the reaction (6, 20). Results for mutants of DHFR (22–25) have been interpreted as showing a central role for protein dynamics in catalysis. However, mutations that affect protein dynamics may actually influence the chemical reaction in other ways (7), such as through changing conformational preferences of the enzyme (26). Strong evidence exists against a direct coupling of large-scale millisecond protein motions to the reaction coordinate during hydride transfer from NADPH to H₂F (6, 7, 27–29), but the coupling of short-range promoting enzyme motions to the reaction coordinate in DHFR cannot be excluded experimentally (6, 22, 27). The effects of protein dynamics on chemical reactions in enzymes have previously been investigated directly only by simulations. These have found that the effects of mutation on reaction in DHFR are not dynamical; rather, the free-energy barrier for reaction is affected (7, 30, 31). Given the lack of clear evidence of dynamical effects on the reaction per se, more direct probes are required.Open in a separate windowFig. 1.Active site of EcDHFR in the reaction-ready configuration. Substrate, cofactor, and key amino acid residues are shown as sticks. The portion of the reactants treated quantum mechanically in the QM/MM simulations (SI Text) is shown in an overlaid surface representation. The figure was created from the crystal structure with PDB code 1RX2, using UCSF Chimera (60).Dynamical effects can be rigorously defined as deviations of phenomenological rate constants, k(T), from the predictions of transition-state theory (TST) (32–34). In a canonical ensemble, phenomenological rate coefficients are typically represented aswhere R is the ideal gas constant, k_B is the Boltzmann constant, h is Planck’s constant, Q^TS and Q^R are the respective transition-state (TS) and reactant (R) partition functions, ε^TS is the classical transition-state barrier height, is the quasiclassical activation free energy (for more detail see SI Text) (35), and Γ(T) is the temperature-dependent transmission coefficient, which generally lumps together the so-called “dynamical” corrections to the classical TST expression. In the limit of classical TST, Γ(T) in Eq. 1 is equal to unity. In such circumstances, an Arrhenius plot of ln(k(T)) vs. 1/T should be nearly linear, as long as the temperature range is small enough that the preexponential factor is approximately constant.Several enzymes show nonlinear Arrhenius plots for H-transfer reactions (5, 36–40). However, the microscopic origin of these nonlinearities remains an open question. The most common explanations invoke recrossing and tunneling, both of which are folded into Γ(T),where the recrossing transmission coefficient, γ, corrects the rate coefficient for trajectories that recross the dividing surface back to the reactant valley, and the tunneling coefficient, κ, accounts for reactive trajectories that do not reach the classical threshold energy. In general, 0 ≤ γ(T) ≤ 1, with values less than unity arising from the coupling of the reaction coordinate to other coordinates (discussed in further detail below). γ(T) can be estimated from molecular dynamics (MD) trajectories starting from the TS with a thermal distribution of velocities. Recent studies on several enzyme-catalyzed reactions (11–14) suggest that recrossing coefficients tend to be somewhat closer to unity than the corresponding counterpart reactions in solution. In general, κ(T) ≥ 1, with values larger than unity when quantum tunneling is important (41, 42).Isotopic substitution of substrates or cofactors has provided strong evidence for quantum tunneling in enzyme reactions. The temperature and pressure dependences of experimentally observed reaction rates and kinetic isotope effects have been interpreted to be a consequence of protein motions on the pico- to femtosecond timescale that reduce the width and/or height of the potential energy barrier along the chemical reaction coordinate (1–4, 43). Others have postulated that millisecond conformational fluctuations may also be involved in driving the chemical step of the reaction (22). To focus more directly on protein dynamics, rather than dynamics of the reactants, entire enzymes can be isotopically substituted, with all nonexchangeable atoms of a particular type (e.g., N, C, H) replaced by a heavier isotope; the “heavy” enzyme can then be compared with its natural, lighter counterpart. Within the Born–Oppenheimer approximation, the electronic potential energy surface, V, governing atomic motion is identical in the light and heavy enzymes. The forces acting on the atoms are also identical, being the negative gradient of the potential with respect to atomic coordinates (i.e., –dV/dq = F, where F is the force acting on an atom and q is a vector of atomic coordinates). Consequently, any differences in reaction rate between the light and heavy enzymes must arise from mass-induced differences in atomic motions, ranging from fast bond vibrations on the femtosecond timescale to conformational changes on the millisecond timescale.Isotopic substitution of HIV protease, purine nucleoside phosphorylase, alanine racemase, and pentaerythritol tetranitrate reductase has been proposed to affect catalysis by changing ultrafast vibrations that couple to the reaction coordinate (44–47). However, the precise manner in which such mass-dependent effects impact the different terms of the preexponential factor in Eq. 1 remains uncertain. Exactly how γ(T) and κ(T) contribute to Γ(T), and in particular how these are affected by protein dynamics, remains a fundamental and hotly debated question with important consequences for understanding enzyme catalysis. Using a combination of experiment, theory, and computation, we analyze dynamical effects by comparing the rate coefficients for hydride transfer in NADPH catalyzed by both “heavy” (¹⁵N, ¹³C, ²H isotopically substituted) and “light” (natural isotopic abundance) EcDHFR. We have measured, analyzed, and simulated the temperature dependence of the EcDHFR-catalyzed hydride transfer from NADPH to H₂F in the heavy and light enzymes. A key component of these experiments is the fact that we isotopically modified only the protein, leaving the substrate unchanged. This universal isotopic substitution of the protein provides an exquisitely sensitive means of probing dynamical effects.     

Qualitative and quantitative study of dihydrofolate reductase in myelodysplastic syndromes

Dihydrofolate reductase (FH2-R) was studied cytochemically in the bone marrow erythroblasts of 20 normal controls and 46 patients with myelodysplastic syndromes (MDSs) classified according to FAB, prior to therapy. The reaction product was quantified for the same samples with a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythroid cells and persisted at high levels in MDS cells. The high level of FH2-R may be related to the malignant transformation of the cells, or to increased compensatory erythropoietic activity of ineffective erythropoiesis, or to both.