The role of protein
dynamics in the reaction catalyzed by dihydrofolate
reductase from the hyperthermophile Thermotoga maritima (TmDHFR) has been examined by enzyme isotope substitution (15N, 13C, 2H). In contrast to all other
enzyme reactions investigated previously, including DHFR from Escherichia coli (EcDHFR), for which isotopic substitution
led to decreased reactivity, the rate constant for the hydride transfer
step is not affected by isotopic substitution of TmDHFR. TmDHFR therefore
appears to lack the coupling of protein motions to the reaction coordinate
that have been identified for EcDHFR catalysis. Clearly, dynamical
coupling is not a universal phenomenon that affects the efficiency
of enzyme catalysis

E. Joel Loveridge (1282176)

Louis
Y. P. Luk (1317813)

Rudolf K. Allemann (1282170)

PubMed

Joel Loveridge

Cronfa at Swansea University

Different Dynamical Effects in Mesophilic and Hyperthermophilic Dihydrofolate Reductases

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Different
Dynamical Effects in Mesophilic and Hyperthermophilic
Dihydrofolate Reductases

鈴木, 健太

Hokkaido University Collection of Scholarly and Academic Papers

昇温熱分解¹⁴C法による堆積物年代推定と北極海イベント層の層位学的研究 [論文内容及び審査の要旨]

Louis Y. P. Luk

E. Joel Loveridge

Rudolf K. Allemann

Crossref

Journal of the American Chemical Society

The role of protein dynamics in the reaction catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) has been examined by enzyme isotope substitution (15N, 13C, 2H). In contrast to all other enzyme reactions investigated previously, including DHFR from Escherichia coli (EcDHFR), for which isotopic substitution led to decreased reactivity, the rate constant for the hydride transfer step is not affected by isotopic substitution of TmDHFR. TmDHFR therefore appears to lack the coupling of protein motions to the reaction coordinate that have been identified for EcDHFR catalysis. Clearly, dynamical coupling is not a universal phenomenon that affects the efficiency of enzyme catalysis

Luk, Louis Y. P.

Loveridge, E. Joel

Allemann, Rudolf K.

Online Research @ Cardiff

Different Dynamical Effects in Mesophilic and HyperthermophilicDihydrofolate ReductasesLouis Y. P. Luk,† E. Joel Loveridge,† and Rudolf K. Allemann*,†,‡†School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom‡Cardiff Catalysis Institute, School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom*S Supporting InformationABSTRACT: The role of protein dynamics in thereaction catalyzed by dihydrofolate reductase from thehyperthermophile Thermotoga maritima (TmDHFR) hasbeen examined by enzyme isotope substitution (15N, 13C,2H). In contrast to all other enzyme reactions investigatedpreviously, including DHFR from Escherichia coli(EcDHFR), for which isotopic substitution led todecreased reactivity, the rate constant for the hydridetransfer step is not affected by isotopic substitution ofTmDHFR. TmDHFR therefore appears to lack thecoupling of protein motions to the reaction coordinatethat have been identified for EcDHFR catalysis. Clearly,dynamical coupling is not a universal phenomenon thataffects the efficiency of enzyme catalysis.Kinetic isotope effect (KIE) studies with isotopically labeledsubstrates are a well-established method to probe themechanisms of enzymatic reactions.1−7 More recently, kineticstudies have been performed where entire enzymes have beenisotopically substituted with all 14N, 12C, and nonexchangeable1H atoms replaced by heavier stable isotopes.8−13 Suchcomplete enzyme isotopic substitution slows protein motionsranging from femtosecond bond vibrations to millisecondstructural changes, while the electrostatic properties areunaffected.14,15 Comparing the kinetic behavior of “heavy”enzymes (isotopically labeled with 15N, 13C, and 2H) with thatof “light” enzymes (with natural isotope abundance) cantherefore reveal information about the relationship betweenenzyme catalysis and protein dynamics.8,9Isotope substitution in dihydrofolate reductase fromEscherichia coli (EcDHFR) and one of its mutants,10,11 purinenucleoside phosphorylase,8 HIV protease,9 alanine racemase,13and pentaerythritol tetranitrate reductase12 causes noticeablechanges in the rates of the chemical steps, demonstrating thatprotein motions have a small but measurable effect on thecatalyzed reactions. DHFR catalyzes the formation oftetrahydrofolate (H4F) by transferring hydride from C4 ofNAPDH to C6 of dihydrofolate (H2F) and adding a proton toN5 of H2F. It has long been used as a model to examine theeffects of protein dynamics on enzyme catalysis.10,11,16−27 Inthe case of EcDHFR, “promoting motions” had beenhypothesized to enhance hydride transfer.28−32 In contrast,combined experimental and computational analyses ofcomplete enzyme isotopic substitution indicated that whileprotein motions do couple to the reaction coordinate, they donot drive tunneling or modulate the barrier of the chemicaltransformation.10,11 Rather, the reactivity difference betweenthe “light” and “heavy” enzymes is due to a change in thefrequency of dynamical recrossing, nonproductive trajectoriesthat do not remain on the product side of the transition-statedividing surface.33 Interestingly, these studies indicated that thedynamical coupling to the chemical step is enhanced in acatalytically compromised mutant.10 Recrossing coefficients forenzyme-catalyzed reactions tend to be closer to unity than fortheir counterparts in solution,34−37 and it has been shown thatcompression of the reaction coordinate can in fact beanticatalytic in enzymes.38 These observations suggest thatefficient enzymes may be characterized by reduced dynamicalcoupling to the reaction coordinate relative to the uncatalyzedreactions.EcDHFR is a relatively flexible monomeric enzyme thatcontains several mobile segments, namely, the M20, FG, andGH loops (Figure 1).40 These loops control the physical stepsof substrate binding and product release by switching theenzyme between the “closed” and “occluded” conformations.40In contrast, DHFR from the hyperthermophile ThermotogaReceived: March 16, 2014Published: April 29, 2014Figure 1. Cartoon representation of (left) TmDHFR (PDB entry1D1G)39 and (right) EcDHFR (PDB entry 1DRE).40 Only onesubunit and the dimer interface of TmDHFR are shown. The ligandsNADPH and methotrexate are shown as sticks. The M20 loop (red) isshown in its closed conformation in EcDHFR and in the openconformation in TmDHFR. The FG and GH loops are highlighted inblue and green, respectively.Communicationpubs.acs.org/JACS© 2014 American Chemical Society 6862 dx.doi.org/10.1021/ja502673h | J. Am. Chem. Soc. 2014, 136, 6862−6865Terms of Use CC-BYmaritima (TmDHFR) forms a very stable dimer, and the FGloop is locked in the dimer interface (Figure 1).39,41 Thesestructural features contribute to the exceptional thermostabilityof TmDHFR (Tm = 83 °C). On the other hand, TmDHFRappears to be fixed in an open conformation and showscatalytic activity considerably lower than that ofEcDHFR.39,41,42 The KIE on the TmDHFR-catalyzed hydridetransfer was found to be highly temperature-dependent below25 °C but largely temperature-independent at elevatedtemperatures.42 The reaction proceeds with a contributionfrom quantum-mechanical tunneling, particularly at lowtemperatures, but this is not promoted by long-range proteinmotions.20−23,43 To investigate whether the environmentalcoupling to hydride transfer observed in EcDHFR10,11 alsoapplies to an enzyme with less conformational flexibility, akinetic comparison of “heavy” TmDHFR with its “light”counterpart was performed.“Heavy” TmDHFR was produced in minimal mediumcontaining only 15N-, 13C-, and 2H-labeled ingredients [seethe Supporting Information (SI)]. Purified “heavy” TmDHFRshowed a molecular weight increase of 10.6%, indicating thatover 98% of the nonexchangeable atoms had been replaced bythe corresponding heavy isotopes (Figure S1 in the SI).Circular dichroism spectra of the “light” and “heavy” enzymeswere essentially superimposable (Figure S2), suggesting thatisotope substitution does not significantly affect the secondarystructure of TmDHFR.The reactivities of “light” and “heavy” TmDHFR were firstcharacterized at pH 7 under steady-state conditions, wherehydride transfer is only partially rate-limiting.42 The steady-state rate constants for “light” TmDHFR, kcatLE, are higher thanthose for its “heavy” counterpart, kcatHE (Figure 2 and Table S1 inthe SI). Between 15 and 65 °C, the magnitude of the enzymeKIEcat (kcatLE/kcatHE ≈ 1.35) is largely unchanged, but it increases to1.73 ± 0.01 at 7 °C (Figure 2 and Table S2). Interestingly, thetemperature dependence of the enzyme KIEcat in TmDHFRgreatly differs from that of the EcDHFR KIEcat, which increasessteadily from 1.04 ± 0.03 at 10 °C to 1.16 ± 0.01 at 35 °C.11The Michaelis constants (KM) of TmDHFR were found tobe mildly temperature-dependent. The KM values for bothNADPH and DHF are ∼1 μM at 45 °C and identical within theexpermental error for “light” and “heavy” TmDHFR (TableS3); they decrease to <0.5 μM at 10 and 20 °C. It is unclearwhether there is a difference between the KM values for “light”and “heavy” TmDHFR at low temperature, since these lowvalues were difficult to measure accurately. Nevertheless, thenature of tight ligand binding in TmDHFR remains unchangedupon enzymatic isotope substitution.42The effect of heavy isotope substitution on the TmDHFR-catalyzed hydride transfer was measured in single-turnoverexperiments. At pH 7.0, the rate constants of the chemical stepfor “light” and “heavy” TmDHFR are essentially identical,giving an enzyme KIEH (kHLE/kHHE) of ∼1 at all temperatures(Figure 2 and Tables S1 and S2). In contrast, for EcDHFR theenzyme KIEH increases from 0.93 ± 0.02 at 10 °C to 1.18 ±0.09 at 40 °C, leading to an activation energy difference (ΔEa)of 5.78 ± 1.61 kJ mol−1.11 The apparent pKa values for hydridetransfer for “light” and “heavy” TmDHFR are also similar(Table S4). As the enzyme KIEH does not change significantlywith pH for either TmDHFR (Figure S3 and Table S4) orEcDHFR,11 the difference in the pKa values of the two enzymesis unlikely to be significant to our discussion, and comparisonof the enzyme KIEH values at a single pH value is appropriate.To the best of our knowledge, TmDHFR is to date the onlyenzyme for which no noticeable “heavy” enzyme KIE has beenobserved for the chemical step.8−13Under steady-state conditions, heavy isotope substitutioncauses a strong reactivity difference for TmDHFR. The absenceof an effect on hydride transfer on the other hand suggests thatprotein motions play a role only in the physical steps duringTmDHFR catalysis. This finding is in agreement with previousinvestigations of the solvent effects, which showed no sign ofFigure 2. Experimental TmDHFR data for steady-state and hydride transfer (pre-steady-state) rate constants at pH 7. (A) Steady-state kinetic data;(B) pre-steady-state kinetic data. Data points and Arrhenius fits are shown for “light” (red circles) and “heavy” (blue triangles) TmDHFR. (C, D)Enzyme KIEs (ratio of rate constants for “light” and “heavy” TmDHFR, kLE/kHE) under steady-state and pre-steady-state conditions, respectively.Journal of the American Chemical Society Communicationdx.doi.org/10.1021/ja502673h | J. Am. Chem. Soc. 2014, 136, 6862−68656863long-range coupled motions.17,19,22 Many TmDHFR variantswith disrupted dimer interfaces show a larger decrease insteady-state turnover than in hydride transfer rate con-stants.20,43 Hence, the reduced kcatHE is likely due to an isotopeeffect on the intra- and/or intersubunit motions that areimportant in the physical steps of catalysis.20,43,44 In addition,the enzyme KIEcat for EcDHFR increases with temperature, butfor the double mutant EcDHFR-N23PP/S148A, KIEcat remainsconstant (∼1),10,11 consistent with the observation that therelease of the product tetrahydrofolate is rate-limiting in thewild-type enzyme and involves a large conformationalchange11,40 while the release of NADP+ is rate-limiting in themutant and most likely involves only a small conformationalchange.10,28 Conformational changes in TmDHFR appear to beminimal.39,43 Hence, the magnitude of the enzyme KIEcat(∼1.35) in TmDHFR is relatively constant at most temper-atures. In turn, the abrupt increase in the enzyme KIEcat at lowtemperatures could be caused by a switch in the conformationalequilibrium favorable for reaction. This needs to be verified byadditional studies, such as binding studies on isotopicallylabeled transition-state analogues and/or protein segments.There has been continuing controversy over the potentialrole of protein motions in “promoting” enzymatic hydrogentunne l ing a t phys io log i c a l l y r e l evan t t empera -tures.10,11,16,28,45−54 On the basis of our previous calculationsperformed on EcDHFR,10,11 the enzyme kinetic isotope effectson hydride transfer (KIEH) reported here imply the absence ofdynamical coupling to the reaction coordinate in TmDHFR atall temperatures examined. We have shown previously that thedynamical coupling to the chemical step is enhanced in acatalytically compromised mutant of EcDHFR.10,11 TmDHFRmay derive a slight benefit from the lack of dynamical coupling.Conformational and structural constraints imposed by dimeri-zation are instead the major cause of TmDHFR’s low activity.In EcDHFR, formation of the closed DHFR−substratecomplex excludes solvent molecules from the active site andallows the formation of a geometric and electrostatic environ-ment conducive to hydride transfer, thus lowering thereorganization energy (the energy required to reorient thereactants during the reaction).40 In TmDHFR, such conforma-tional sampling is prevented by the enzyme’s dimeric structure,generating a DHFR−substrate complex in which the active siteis exposed to solvent interactions. This compromises theelectrostatic preorganization (the enzyme’s ability to arrangethe substrates with “product-like” geometry and electrostatics),leading to an increase in the reorganization energy and arelatively low rate constant for hydride transfer.In summary, the enzyme KIEH of ∼1 observed for TmDHFRreveals much information about the structural and dynamicalproperties of the enzyme. If the enzyme KIE reports onrecrossing events in TmDHFR in the same way as it does inEcDHFR,10,11 then it appears that recrossing events inTmDHFR are unaffected by protein dynamics. Previous studiesof EcDHFR and its variant indicated that dynamical effectscontribute only a small change to the activation free energy.10,11Therefore, the low activity of TmDHFR is most likely due topoor electrostatic preorganization and is unrelated to dynamicalcoupling. The inability of TmDHFR to form a closedconformation favorable for reaction outweighs any potentialsmall benefit from the reduction in recrossing events. Thesecharacteristics of TmDHFR may reflect an evolutionary trade-off between catalytic activity and thermal stability. Therelationship between dynamics and barrier crossing/recrossingmust be examined further by experimentation and calculation.■ ASSOCIATED CONTENT*S Supporting InformationFull experimental procedures; mass spectra of purified proteins;circular dichroism spectra; tabulated experimental data for kH,kcat, and enzyme KIEs; and pH dependence of kH. This materialis available free of charge via the Internet at http://pubs.acs.org.■ AUTHOR INFORMATIONCorresponding Authorallemannrk@cf.ac.ukNotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThis work was supported by Grant BB/J005266/1 (R.K.A.)from the UK Biotechnology and Biological Sciences ResearchCouncil (BBSRC). The authors express their gratitude to IñakiTuñoń and Vicent Moliner for their insightful comments onthe manuscript.■ REFERENCES(1) Schramm, V. L. Acc. Chem. 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Different dynamical effects in mesophilic and hyperthermophilic dihydrofolate reductases

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昇温熱分解¹⁴C法による堆積物年代推定と北極海イベント層の層位学的研究 [論文内容及び審査の要旨]

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Hokkaido University Collection of Scholarly and Academic Papers

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