Báo cáo khoa học: The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex ppt

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The pH dependence of kinetic isotope effects inmonoamine oxidase A indicates stabilization of theneutral amine in the enzyme–substrate complexRachel V. Dunn1, Ker R. Marshall2, Andrew W. Munro1and Nigel S. Scrutton11 Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, UK2 Department of Biochemistry, University of Leicester, UKThe mammalian monoamine oxidases (MAO) (EC1.4.3.4) are flavoproteins localized to the outer mito-chondrial membrane, and contain a FAD cofactorcovalently linked via the 8a-methyl group to an activesite cysteine residue [1]. They catalyse the oxidativedeamination of neurotransmitters (e.g. dopamine andserotonin) and exogenous alkylamines, and are there-fore important pharmaceutical targets for the develop-ment of antidepressants and neuroprotective agents [2].The catalytic cycle for monoamine oxidase activity isshown in Scheme 1.A number of mechanisms for MAO-catalysed amineoxidation have been proposed over the years, and sev-eral reviews are available [3–5]. There are currentlyKeywordskinetic isotope effect; mechanism;monoamine oxidase; pH dependenceCorrespondenceN. S. Scrutton, Faculty of Life Sciences,Manchester Interdisciplinary Biocentre,University of Manchester, 131 PrincessStreet, Manchester M1 7DN, UKFax: +44 161 3068918Tel: +44 161 3065152E-mail: nigel.scrutton@manchester.ac.uk(Received 10 April 2008, revised 25 May2008, accepted 2 June 2008)doi:10.1111/j.1742-4658.2008.06532.xA common feature of all the proposed mechanisms for monoamine oxidaseis the initiation of catalysis with the deprotonated form of the amine sub-strate in the enzyme–substrate complex. However, recent steady-statekinetic studies on the pH dependence of monoamine oxidase led to the sug-gestion that it is the protonated form of the amine substrate that binds tothe enzyme. To investigate this further, the pH dependence of monoamineoxidase A was characterized by both steady-state and stopped-flow tech-niques with protiated and deuterated substrates. For all substrates used,there is a macroscopic ionization in the enzyme–substrate complex attrib-uted to a deprotonation event required for optimal catalysis with a pKaof7.4–8.4. In stopped-flow assays, the pH dependence of the kinetic isotopeeffect decreases from approximately 13 to 8 with increasing pH, leading toassignment of this catalytically important deprotonation to that of thebound amine substrate. The acid limb of the bell-shaped pH profile for therate of flavin reduction over the substrate binding constant (kred⁄ Ks, report-ing on ionizations in the free enzyme and ⁄ or free substrate) is due todeprotonation of the free substrate, and the alkaline limb is due to unfa-vourable deprotonation of an unknown group on the enzyme at high pH.The pKaof the free amine is above 9.3 for all substrates, and is greatly per-turbed (DpKa$ 2) on binding to the enzyme active site. This perturbationof the substrate amine pKaon binding to the enzyme has been observedwith other amine oxidases, and likely identifies a common mechanism forincreasing the effective concentration of the neutral form of the substratein the enzyme–substrate complex, thus enabling efficient functioning ofthese enzymes at physiologically relevant pH.AbbreviationsES, enzyme–substrate; KIE, kinetic isotope effect; MAO, monoamine oxidase; PEA, phenylethylamine; TMADH, trimethylaminedehydrogenase.3850 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBSthree main mechanistic proposals for MAO catalysis.These comprise: (a) the concerted polar nucleophilicmechanism; (b) the direct hydride transfer mechanism;and (c) the single electron transfer mechanism. Recentsupport for the concerted polar nucleophilic mecha-nism has come from kinetic and structural studies ontyrosine mutants of MAO B [6], and also from compu-tational studies [7,8]. However, analysis of nitrogenisotope effects conducted on a related amine oxidase,N-methyltryptophan oxidase, supported either a directhydride transfer mechanism or, possibly, a discreteelectron transfer mechanism [9]. Support for a modi-fied single electron transfer mechanism came followingthe identification of a stable tyrosyl radical in partiallyreduced MAO A [10]. More recent EPR studies havequestioned this assignment and suggested that the rad-ical species detected in partially reduced MAO is duesolely to the covalently linked flavin semiquinone,leading to support for the direct hydride transfermechanism [11].A common feature of all the proposed mechanismsis the initiation of catalysis with the deprotonatedform of the amine substrate, and it is widely acceptedthat it is the deprotonated form of the substrate thatbinds in the functional enzyme–substrate (ES) com-plex [12–14]. By contrast, recent kinetic studies onthe pH dependence of the steady-state kinetic param-eters for MAO A were interpreted to indicate that itis the protonated form of the substrate that binds tothe enzyme [15]. Due to the conflicting evidence fromthe relatively few studies on the effects of pH onMAO catalysis, a more comprehensive analysis isrequired.The present study reports on the pH dependence ofrecombinant human liver MAO A as characterized byboth steady-state and stopped-flow techniques. Theeffect of pH on the kinetic isotope effect (KIE) of thereductive half-reaction is also presented. The resultsobtained provide insight into how monoamine oxidasesare able to function efficiently at physiological pH withthe deprotonated amine substrate, despite the high pKavalues of common substrates.Results and DiscussionCatalytically influential macroscopic ionizationsThe pH dependence of the catalytic rate was studiedby both stopped-flow and steady-state techniques.Although the catalytic activity of MAO A has beenshown to be dominated by the reductive half-reaction,this may change with pH, leading to a different pHdependence for the reductive half reaction comparedto complete catalytic turnover. Also, a range of sub-strates were analysed to establish whether the observedkinetic trends were applicable for all amine substrates.For example, although benzylamine is a well character-ized substrate for MAO A, all naturally occurring sub-strates contain an ethylamine group in the structure.All steady-state kinetic measurements were per-formed in air-saturated buffers, which have beenshown to saturate the enzyme with the second sub-strate, oxygen [16]. The kcatvalues for benzylamine(see supplementary Fig. S1) and kynuramine exhibit asigmoidal dependence upon pH, as shown in Fig. 1Afor kynuramine, indicating the presence of a singlemacroscopic ionization with a pKavalue of 7.9 ± 0.1obtained from curve fitting for both substrates. Theobserved macroscopic ionization corresponds to agroup in the ES complex that must be deprotonatedfor optimal activity. The kcat⁄ Kmvalues exhibit a bell-shaped pH profile with corresponding pKavalues of8.5 ± 0.1 and 9.2 ± 0.1 for benzylamine (see supple-mentary Fig. S1), and 8.0 ± 0.2 and 8.8 ± 0.2 forkynuramine (Fig. 1B). These results indicate that, withincreasing pH, a favourable deprotonation step isfollowed by an unfavourable deprotonation event,either in the free enzyme or free substrate, to producethe bell-shaped pH profile.At pH 7.5 and below, the flavin monitored reductivehalf-reaction transients from stopped-flow assays werefitted using a single exponential function to determinethe apparent rate constants for FAD reduction. How-ever, above pH 7.5, the reaction traces were fittedinstead with a double-exponential function, as a second,slower process was resolved in the flavin reductive reac-tion. This biphasic behaviour has been observed previ-ously with para-substitued phenylethylamines, and theslow phase was attributed to the release of the imineproduct from the reduced enzyme [17]. Because the slowphase was only a minor component of the total ampli-tude change (20–30% at most) and did not vary withsubstrate concentration, only the substrate dependenceof the fast phase was analysed further. As expected, thepH dependence of the kinetic parameters for the reduc-tive half-reaction of benzylamine oxidation exhibitedScheme 1. Catalytic cycle of monoamine oxidase.R. V. Dunn et al. Isotope effects and their pH dependence in MAO AFEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3851similar pH profiles to those obtained for the equivalentsteady-state parameters (see supplementary Fig. S2). Ateach pH, the value of kredwas found to be less thanthat of kcat, which has been observed previously inkinetic studies with MAO A [12]. This was attributed toaggregation of the detergent solubilized enzyme at thehigh concentrations required for stopped-flow assays.To minimize this potential effect, the same concentra-tion of MAO A was used in all stopped-flow experi-ments. The kredexhibited a single ionization with acorresponding pKaof 7.4 ± 0.1, and the kred⁄ Ksexhib-ited a bell-shaped profile with pKavalues of 8.6 ± 0.7and 8.3 ± 0.7 obtained from curve fitting.By contrast to all other substrates, the pH depen-dence of kredfor MAO A-catalysed phenylethylamine(PEA) oxidation displayed a bell-shaped profile, withcorresponding pKavalues of 8.4 ± 0.2 and 8.7 ± 0.2(Fig. 1C). The cause of the additional macroscopic ioni-zation on the alkaline side of the pH profile for PEA isunknown. For benzylamine and PEA, it has been estab-lished that the rate-limiting step of flavin reduction isdue to aC-H bond cleavage, and it is unlikely that a dis-tinct catalytic step affects flavin reduction to producethe different pH dependence. From quantitative struc-ture activity studies with MAO A, it has been shownthat different factors influence the correct positioningof para-substituted phenylethylamines compared topara-substituted benzylamines, and that these arerequired for efficient catalysis [12,17]. It was suggestedthat the greater steric flexibility of the ethylamine sidechain allows efficient aC-H bond cleavage without con-fining the phenyl ring to a specific orientation. There-fore, the greater flexibility of the substrate when boundto the active site may allow it to contact additionalionizable residues that influence the correct orientationfor catalysis and affect the resulting pH profile. Thekred⁄ Ksdata also exhibit a bell-shaped pH profile, butmeaningful pKavalues cannot be determined due to thelarge errors associated with these data (Fig. 1D). Asummary of all pKavalues is given in Table 1.pH dependence of KIEs identifies substrateionization in the ES complexAs the amine substrates are able to ionize over the pHrange investigated, some of the observed macroscopic0. (s–1)pH01020304050kcat/Km (s–1 mM–1)pH0. (s–1)pH6.06.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.06.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0-20020406080100120140kred/Ks (s–1 mM–1)pHFig. 1. (A, B) pH dependence of the steady-state kinetic parameters of MAO A-cataly-sed oxidation of kynuramine at 20 °C. (C, D)pH dependence of the reductive half-reac-tion of MAO A-catalysed oxidation of phen-ylethylamine at 20 °C.Table 1. pKavalues obtained from curve fitting for MAO A at 20 °C. ND, not determined.Substrate MethodES complex Free E or SpKa1pKa2pKa1pKa2Benzylamine Steady-state 7.9 ± 0.1 – 8.5 ± 0.1 9.2 ± 0.1Kynuramine Steady-state 7.9 ± 0.1 – 8.0 ± 0.2 8.8 ± 0.2Benzylamine Stopped-flow 7.4 ± 0.1 – 8.6 ± 0.7 8.3 ± 0.7PEA Stopped-flow 8.4 ± 0.2 8.7 ± 0.2 ND NDIsotope effects and their pH dependence in MAO A R. V. Dunn et al.3852 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBSionizations may be due to the substrate rather than togroups on the enzyme. A potential way to identify sub-strate ionizations is to perturb the substrate pKa(e.g.by deuteration) and to observe a corresponding shiftin the macroscopic ionization. Deuteration of theatoms bonded to the amine nitrogen is known to causean increase in the amine pKa; in part due to: (a) theshorter C-D bond length leading to a greater chargedensity on the carbon and hence greater nitrogen lonepair availability and (b) the greater reduced mass ofthe deuterated analogue for the N-H stretching fre-quency, causing it to lie lower in the asymmetricpotential energy well (lower zero point energy) relativeto the protiated substrate [18–20]. The pH dependenceof the reductive half-reaction of benzylamine oxidationwas determined at a saturating substrate concentrationof 5 mm, for both the protiated and deuterated forms.The pH profile of kredfor both substrates is shown inFig. 2, and a small alkaline shift is observed for deu-terated benzylamine relative to protiated benzylamine,which results in a decrease of the calculated KIE from13 to 8 with increasing pH (Fig. 2, inset). A similareffect has been seen in studies with trimethylaminedehydrogenase (TMADH), where substrate perdeutera-tion caused a shift in the observed macroscopic ioniza-tion in the ES complex, resulting in a strongdependence of the KIE upon pH [21]. This result, com-bined with mutagenesis work on TMADH, led to theassignment of the ionization as that of bound sub-strate. It is likely that a similar effect is observed withMAO A, where the observed macroscopic ionization isdue to deprotonation of the bound amine substrate.The effect of substrate deuteration was more signifi-cant for TMADH, and may be explained by thegreater increase in substrate pKaupon perdeuterationof trimethylamine (DpKa= 0.3) [18] compared to a-Cdeuteration of benzylamine (expected DpKa= 0.032)[19]. A mechanism describing the ionization of the sub-strate and its effect on flavin reduction is shown inScheme 2, where KASand KAESare the dissociationconstants for the free substrate and the enzyme-boundsubstrate, respectively [22]. It is assumed that the rateof flavin reduction (kred) is slow relative to the dissoci-ation steps, so that they remain in thermodynamicequilibrium. It can be seen that if the pKaof the aminesubstrate is increased (e.g. in the deuterated analogue),this will lead to a greater proportion of the unreactiveESH+form relative to ES at a given pH. Therefore,the observed KIE will appear inflated at low pH, andbe greater than that due purely to bond breakageeffects.Perturbation of amine pKamechanism ofmonoamine oxidaseThe accuracy of the derived pKavalues from the bell-shaped pH profiles for kred⁄ Ksor kcat⁄ Kmis quite low.This is partly due to the error associated with fittingthe particular functions to narrow plots because thewidth of the curve is relatively insensitive to the differ-ence in pKavalues when pKa1)pKa2is < 0.6 [22].Despite this drawback, the pH profiles are still of qual-itative value. Based upon the assignment of the ioniza-tion in the ES complex to that of the bound substrate,it follows that the acid limb of the bell-shaped kred⁄ Ksor kcat⁄ Kmprofile is due to deprotonation of the freesubstrate and that the alkaline limb is due to the unfa-vourable deprotonation of an unknown group on theenzyme at high pH. The stated pKavalues of the freesubstrates (9.3–9.9) are higher than those obtainedfrom curve fitting (8.0–8.6) and may simply reflect theerror in curve fitting as mentioned above. The maineffect of substrate binding is to perturb the amine pKato more acidic values; as the bound substrate has apKaof 7.4–8.4, this corresponds to a DpKaof approxi-mately 2 relative to the free substrate. Such an effecthas been seen with other amine oxidases. For example,6 7 8 9 10 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 pH kred (s–1)0.000 0.002 0.004 0.006 0.008 0.010 0.012 6.5 7.0 7.5 8.0 8.5 9.0 9.5681012141618KIEpHkred (s–1)Fig. 2. pH dependence of the reductive half-reaction of MAO A-ca-talysed oxidation at 20 °C with 5 mM benzylamine (filled circles, leftaxis) or 5 mM deuterated benzylamine (open circles, right axis).Inset: calculated KIE as a function of pH.ES + SHk1k1k2k2''KAKASESkred+ESH+E + S ES E + PScheme 2. Control of flavin reduction by substrate ionization.R. V. Dunn et al. Isotope effects and their pH dependence in MAO AFEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3853trimethylamine dehydrogenase, mouse polyamine oxi-dase and monomeric sarcosine oxidase exhibit acidicshifts in substrate pKavalues of 3.3–3.6, 0.8 and 2.6,respectively, upon substrate binding to the active site[18,23,24]. Therefore, the active site of each of theseenzymes is organized to stabilize the neutral form ofthe amine substrate by approximately 11 kJÆmol)1rela-tive to the charged, protonated form.The steady-state oxidation of kynuramine byMAO A has been studied previously, and the overalltrends of the data are very similar to those reported inthe present study, which suggests that variations inbuffer composition have minimal effect [15]. However,the interpretation of the results was different in theprevious study. It was suggested that, due to the initialincrease in rate with increasing pH and the relativeinvariance of the Kmvalues over the same pH range(in which the concentration of the neutral form of thesubstrate would be insignificant compared to the pro-tonated form), it must be the protonated form of thesubstrate that binds to the enzyme, with subsequentsubstrate deprotonation required for catalysis to pro-ceed. Therefore, the macroscopic ionization in the EScomplex was assigned to a group on the enzyme,rather than to the ionization of bound substrate, asindicated by data reported in the present study. Theinvariance of the Kmvalues at low pH makes this aplausible explanation, although it may be over simplis-tic to assume that an exponential dependence of Kmwith pH is required to indicate the binding of thedeprotonated form because the pH dependence of Kmor Ksis affected by all macroscopic ionizations occur-ring in the system [22]. The variations in the Kmor Ksvalues with pH for all substrates used in the presentstudy are shown in Table 2. Unlike the values forkynuramine, the Kmor Ksvalues for all othersubstrates tested exhibited a general decrease withincreasing pH in the range from $ 6.5–8.5. When thepKmor pKsvalues are plotted as a function of pH(results not shown), the initial slope at low pH is < 1,which may simply reflect that the relevant macroscopicionizations are not sufficiently separated to be individ-ually identified. There are too few points at high pHto accurately calculate the change of slope that occursabove pH 9 for all substrates, although it is clear thatthe Kmand Ksvalues are increased.ConclusionsDespite the suggestion that it is the protonated formof the substrate that binds the enzyme, it is difficult toenvisage specific binding of the charged substrate whenthe active site is organized for binding and activationof the neutral form. In addition, the pH dependence ofthe KIE and the observation of similar perturbationeffects on substrate pKavalues with other amine oxid-ases further support the catalytic significance of thedeprotonated form. Thus, we propose that binding ofthe substrate to the active site leads to a perturbationof the pKa, effectively increasing the concentration ofthe neutral amine species. We do not propose that it isonly the protonated form that initially binds, butrather that preferential binding of the deprotonatedform to the active site leads to a shift in the equilib-rium of the substrate ionization. The present studyemphasizes the benefits of using deuteration of com-pounds in conjunction with standard stopped-flow andsteady-state analyses to provide deeper insight intoreaction mechanism. In the case of the amine oxidases,the perturbation of the substrate pKaupon binding tothe active site appears to be a general feature, allowingefficient function of the enzyme at physiologicallyrelevant pH values.The crystal structure of MAO A has recently beensolved to 2.2 A˚resolution [25], allowing a moredetailed knowledge of the active site geometry ofTable 2. pH dependence of Kmand Ksvalues determined for MAO A at 20 °C.pHKm(mM) Ks(mM)Benzylamine Kynuramine Benzylamine PEA6.5 – 0.042 ± 0.003 0.213 ± 0.012 0.019 ± 0.0177.0 0.31 ± 0.04 0.047 ± 0.003 0.134 ± 0.032 0.145 ± 0.0077.2 – – 0.136 ± 0.007 –7.5 0.20 ± 0.02 0.041 ± 0.002 0.138 ± 0.014 0.122 ± 0.0158.0 0.10 ± 0.01 0.044 ± 0.003 0.034 ± 0.004 0.078 ± 0.0038.5 0.077 ± 0.002 0.045 ± 0.002 0.033 ± 0.004 0.048 ± 0.0029.0 0.087 ± 0.004 0.082 ± 0.005 0.039 ± 0.004 0.028 ± 0.0029.2 – – 0.079 ± 0.019 0.042 ± 0.0169.5 0.137 ± 0.003 0.261 ± 0.028 0.424 ± 0.108 0.268 ± 0.125Isotope effects and their pH dependence in MAO A R. V. Dunn et al.3854 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBSMAO A. Inspection of the active site suggests thatthere are several candidates responsible for the unfa-vourable deprotonation event that occurs at alkalinepH, including multiple tyrosine residues, the covalentlylinked FAD, and possibly Lys305 that is co-ordinatedto the flavin via a water molecule. To be confidentabout any assignment, future work combining muta-genesis studies with stopped-flow kinetic analysis isrequired.Experimental proceduresMaterialsBis-Tris propane buffer, reduced Triton X-100, kynur-amine, benzylamine, and b-phenylethylamine were obtainedfrom Sigma (St Louis, MO, USA). Deuterated benzylamineHCl (C6D5CD2NH2, 99.2 atom % D) was obtained fromCDN Isotopes (Quebec, Canada).Expression and purification of MAO AThe gene encoding human liver MAO A was amplifiedfrom a cDNA clone obtained from MRC Geneservices(Cambridge, UK) using the primers 5¢-GTCTTCGAAACCATGGAGAATCAAGAGAAGGCGAGTATCGCGGG-3¢ and 5¢-GAGAGCTCGAGAACAGAACTTCAAGACCGTGGCAGGAGC-3¢. The NcoI and Xho I sites used forfurther cloning are shown underlined. The amplified DNAwas first cloned into pGem-T Easy (Promega, Madison,WI, USA) following A-tailing using standard techniques. Amodified version of the pPICZA plasmid (Invitrogen, Carls-bad, CA, USA) was used as the final expression vector, inwhich the NcoI site upstream of the Zeocin resistance genewas mutated using the primer 5¢-GGTGAGGAACTAAAACATGGCCAAGTTGACCAGTGC-3¢ and itsreverse complement. A unique NcoI site was then intro-duced at the multiple cloning site generating a Kozaksequence to allow efficient translation initiation of theinserted gene, using the primer 5¢-CAACTAATTATTCGAAACCATGGATTCACGTGGCCC-3¢ and its reversecomplement. The modified pPICZA vector was thendigested with NcoI and XhoI, and similarly digested maoAinserted following gel purification. The sequence of thecloned gene was confirmed by DNA sequencing. All site-directed mutagenesis reactions were performed using theStratagene QuikChange site-directed mutagenesis kit (Strat-gene, La Jolla, CA, USA) with Pfu Turbo DNA polymer-ase; except that the DNA was transformed into Novabluecompetent cells (Novagen, Madison, WI, USA). ThepPICZAmaoA plasmid was linearized with PmeI and trans-formed into Pichia pastoris strain KM17H by electropora-tion following standard protocols [26]. Successfultransformants were selected on agar plates containing100 lgÆmL)1of Zeocin. Multiple integrants were selectedby growth on plates with increasing Zeocin concentrations,and screened for MAO A expression. Typically, 8 L of cul-ture were grown in baffled flasks in an orbital incubator at30 °C. The cells were harvested for 48 h after methanolinduction by centrifugation at 2000 g for 10 min. The cellswere resuspended in Pichia breakage buffer to approxi-mately 200 gÆL)1and then lysed by passing twice through acell disruptor at 40 000 psi (TS-series 1.1 kW model; Con-stant Systems Ltd, Daventry, UK) followed by cooling onice. MAO A was then purified essentially as described pre-viously [27]. Active fractions eluted from the DEAE-Sepha-rose column were concentrated by ultrafiltration and storedat )80 °C. Prior to use, the enzyme was dialysed extensivelyagainst 20 mm potassium phosphate (pH 7.0), containing20% glycerol, to remove the competitive inhibitor d-amp-hetamine that is present during the later stages of purifica-tion. Typical yields from an 8 L growth were between80–120 mg of purified MAO A. Enzyme concentrationwas determined using an extinction coefficient of12 000 m)1Æcm)1at 456 nm [27].Enzyme assaysRoutine activity measurements were conducted using acontinuous spectrophotometric assay with kynuramine assubstrate. Assays were performed at 25 °Cin50mmpotassium phosphate (pH 7.5), containing 0.5% (w ⁄ v)Triton X-100 and 0.2 mm kynuramine. The activity wascalculated by following the initial increase of A316due toproduction of 4-hydroxyquinone and using an extinctioncoefficient of 12 000 m)1Æcm)1[28]. One unit of enzymeactivity is defined as the amount of enzyme required tooxidize 1 l mol of kynuramine in 1 min.Steady-state kinetic measurementsSteady-state kinetic measurements were performed at 20 °Cin 20 mm Bis-Tris propane buffer containing 0.5% (w ⁄ v)reduced Triton X-100, 50 mm NaCl and 20% glycerol. ThepH of the buffer was set by the addition of small amountsof concentrated HCl or NaOH, and was in the range 6.5–9.5. The rate of enzymatic activity was determined by moni-toring the initial linear increase in absorbance at 250 nmdue to the production of benzaldehyde, employing anextinction coefficient 12 800 m)1Æcm)1[29], and using aVarian Cary 50 Bio spectrophotometer (Varian Inc., PaloAlto, CA, USA). The concentration of benzylamine wastypically in the range 0.02–2 m m, and the assay was startedby the addition of MAO A to a final concentration of0.6 lm. Michaelis–Menten kinetic behaviour was observedat each pH studied; the only exception was at pH 6.5,where the rate of enzymatic activity was only assayed atsaturating substrate concentrations due to the slow rateR. V. Dunn et al. Isotope effects and their pH dependence in MAO AFEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3855observed. Identical experiments were performed with kynur-amine as substrate, but the reaction was monitored at A316,as described above, and the final concentration of MAO Ain the assay was 0.1 lm.Single wavelength anaerobic stopped-flowkinetic experimentsThe reductive half-reaction of MAO A was studied usingan Applied Photophysics SX.18MV stopped-flow spectro-photometer (Applied Photophysics Ltd, Leatherhead, UK)housed in a Belle Technology glove box (< 5 p.p.m.oxygen) (Belle Technology, Portesham, UK). Studies wereperformed in 20 mm Bis-Tris propane buffer containing0.5% (w ⁄ v) Triton X-100, 50 mm NaCl and 20% glycerol.Buffer solutions were purged with nitrogen for 1 h andthen left to equilibrate overnight in the glove box. Dialy-sed MAO A was exchanged into the appropriate anaerobicbuffer by gel exclusion chromatography, and stocksolutions of the substrates were also diluted into theappropriate buffer. To remove any final traces of oxygen,10 units of glucose oxidase (Sigma) and 10 mm glucosewere added per mL of solution and left to incubate for30 min once loaded into the stopped-flow syringes. Thereactions were started by rapid mixing of 10 lm oxidizedMAO A with various concentrations of either benzylamineor phenylethylamine. A minimum of six substrate concen-trations were used at each pH that spanned almost twoorders of magnitude. The rate of flavin reduction wasmonitored under pseudo first-order conditions by follow-ing the decrease in A456.Data analysisSteady-state kinetic data were fitted with the Michaelis–Menten equation using nonlinear least-squares analysisincorporated into the origin software package (OriginLabCorp., Northampton, MA, USA), and the maximal cata-lytic centre activity (kcat) and the Michaelis constant (Km)determined. The observed rates from stopped-flow datawere obtained by fitting the reaction traces to an equationfor either single- or double-exponential decay with offset,as appropriate. Analysis was performed by nonlinear least-squares regression on an Acorn RISC PC (Acorn Com-puters, Cambridge, UK) using spectrakinetics software(Applied Photophysics). The observed rate of enzymereduction was found to have a hyperbolic dependence withrespect to substrate concentration at each pH. The limitingrate of flavin reduction (kred) and the substrate binding con-stant (Ks) were determined as described by Strickland et al.[30] using the origin software package. The pH dependenceof the kinetic parameters were fitted to an equation descri-bing either a single (Eqn 1) or double (Eqn 2) ionization, asappropriate, to obtain the corresponding macroscopic pKavalues.y ¼ EH  10ðÀpHÞþ E  10ðÀpKaÞ10ðÀpHÞþ 10ðÀpKaÞð1Þy ¼Tmax1 þ 10ðpKa1ÀpHÞþ 10ðpHÀpKa2Þð2ÞWhere EH and E are the limiting catalytic activities of theprotonated and deprotonated forms of the ionizationgroup, respectively; and Tmaxis the theoretical maximalvalue. For the pH profile in which a double ionization isobserved, it is assumed that the observed parameter isdependent upon the singly protonated species, thereforeproducing a bell-shaped profile tending towards zero at theextremes of pH. Examples of the reaction transients andfurther details regarding treatment of the data are given insupplementary Figs S3–S5.AcknowledgementsThis work was funded by the UK Biotechnology andBiological Sciences Research Council. 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Proc NatlAcad Sci USA 105, 5739–5744.26 Invitrogen Corporation (1998) EasySelect PichiaExpression Kit: A Manual of Methods For Expressionof Recombinant Proteins Using pPICZ and pPICZa inPichia pastoris. Invitrogen Corporation, San Diego,CA.27 Li M, Hubalek F, Newton-Vinson P & Edmondson DE(2002) High-level expression of human liver monoamineoxidase a in Pichia pastoris: comparison with theenzyme expressed in Saccharomyces cerevisiae. ProteinExpr Purif 24, 152–162.28 Weyler W & Salach JI (1985) Purification and proper-ties of mitochondrial monoamine-oxidase type-A fromhuman-placenta. J Biol Chem 260, 13199–13207.29 Walker MC & Edmondson DE (1994) Structure-activ-ity-relationships in the oxidation of benzylamineanalogs by bovine liver mitochondrial monoamine-oxidase-B. Biochemistry 33, 7088–7098.30 Strickland S, Palmer G & Massey V (1975) Determina-tion of dissociation-constants and specific rate ofconstants of enzyme-substrate (or protein-ligand) inter-actions from rapid reaction kinetic data. J Biol Chem250, 4048–4052.Supplementary materialThe following supplementary material is availableonline:Fig. S1. pH dependence of the steady-state kineticparameters of MAO A-catalysed oxidation of benzyl-amine at 20 °C.Fig. S2. pH dependence of the reductive half-reactionof MAO A-catalysed oxidation of benzylamine at20 °C.Fig. S3. Reaction transient for MAO A-catalysedoxidation of 0.5 mm PEA at pH 9.0 and 20 °C.Fig. S4. Reaction transient for MAO A-catalysedoxidation of 0.4 mm benzylamine at pH 8.5 and20 °C.R. V. Dunn et al. Isotope effects and their pH dependence in MAO AFEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS 3857Fig. S5. Substrate dependence of the reductive half-reaction of MAO A-catalysed oxidation of PEA atpH 8.5 and 20 °C.This material is available as part of the online articlefrom http://www.blackwell-synergy.comPlease note: Wiley-Blackwell is not responsible forthe content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to the corre-sponding author for the article.Isotope effects and their pH dependence in MAO A R. V. Dunn et al.3858 FEBS Journal 275 (2008) 3850–3858 ª 2008 The Authors Journal compilation ª 2008 FEBS . The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex Rachel. PaloAlto, CA, USA). The concentration of benzylamine wastypically in the range 0.02–2 m m, and the assay was startedby the addition of MAO A to a final
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Xem thêm: Báo cáo khoa học: The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex ppt, Báo cáo khoa học: The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex ppt, Báo cáo khoa học: The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex ppt