Báo cáo khoa học: ATP-dependent ligases in trypanothione biosynthesis – kinetics of catalysis and inhibition by phosphinic acid pseudopeptides doc

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ATP-dependent ligases in trypanothionebiosynthesis kinetics of catalysis and inhibitionby phosphinic acid pseudopeptidesSandra L. Oza1, Shoujun Chen2, Susan Wyllie1,James K. Coward2and Alan H. Fairlamb11 Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, UK2 Departments of Medicinal Chemistry and Chemistry, University of Michigan, Ann Arbor, MI, USAChagas’ disease, African sleeping sickness and leish-maniasis (cutaneous, mucocutaneous and visceral) areneglected diseases afflicting millions of people world-wide. All of the drugs used to treat these neglected dis-eases suffer from deficiencies such as poor efficacy,drug resistance, toxicity or high cost of treatment [1].The parasitic protozoa causing these diseases belong tothe order Kinetoplastida, and comparative genomicand biochemical studies have revealed a number ofunique metabolic pathways that are being exploitedfor drug discovery [2]. One of these involves trypano-thione [N1,N8-bis(glutathionyl)spermidine] and trypa-nothione reductase, which replaces not onlyglutathione ⁄ glutathione reductase but also thioredoxin ⁄thioredoxin reductase in mammalian cells [3]. Togetherwith the type I and II tryparedoxin peroxidases [4–6],trypanothione is pivotal in defence against oxidativestress induced by host cell defence mechanisms [7–9] orKeywordsdrug discovery; enzyme mechanism;glutathionylspermidine synthetase; slow-binding inhibition; trypanothione synthetaseCorrespondenceA. H. Fairlamb, Division of BiologicalChemistry and Drug Discovery, WellcomeTrust Biocentre, College of Life Sciences,University of Dundee, Dundee DD1 5EH,UKFax: +44 1382 38 5542Tel: +44 1382 38 5155E-mail: a.h.fairlamb@dundee.ac.ukWebsite: http://www.lifesci.dundee.ac.uk/people/alan_fairlamb/Re-use of this article is permitted inaccordance with the Creative CommonsDeed, Attribution 2.5, which does notpermit commercial exploitation(Received 8 July 2008, revised 4 September2008, accepted 5 September 2008)doi:10.1111/j.1742-4658.2008.06670.xGlutathionylspermidine is an intermediate formed in the biosynthesis oftrypanothione, an essential metabolite in defence against chemical and oxi-dative stress in the Kinetoplastida. The kinetic mechanism for glutathionyl-spermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS)obeys a rapid equilibrium random ter-ter model with kinetic constantsKGSH= 609 lm, KSpd= 157 lm and KATP= 215 lm. Phosphonate andphosphinate analogues of glutathionylspermidine, previously shown to bepotent inhibitors of GspS from Escherichia coli, are equally potent againstCfGspS. The tetrahedral phosphonate acts as a simple ground state ana-logue of glutathione (GSH) (Ki$ 156 lm), whereas the phosphinatebehaves as a stable mimic of the postulated unstable tetrahedral intermedi-ate. Kinetic studies showed that the phosphinate behaves as a slow-bindingbisubstrate inhibitor [competitive with respect to GSH and spermidine(Spd)] with rate constants k3(on rate) = 6.98 · 104m)1Æs)1and k4(offrate) = 1.3 · 10)3s)1, providing a dissociation constant Ki= 18.6 nm.The phosphinate analogue also inhibited recombinant trypanothione syn-thetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosomacruzi and Trypanosoma brucei with Kiappvalues 20–40-fold greater thanthat of CfGspS. This phosphinate analogue remains the most potentenzyme inhibitor identified to date, and represents a good starting pointfor drug discovery for trypanosomiasis and leishmaniasis.OnlineOpen: This article is available free online at www.blackwell-synergy.com AbbreviationsCfGspS, Crithidia fasciculata glutathionylspermidine synthetase; CfTryS, Crithidia fasciculata trypanothione synthetase; EcGspS,Escherichia coli glutathionylspermidine synthetase; GSH, glutathione; GspA, glutathionylspermidine amidase; GspS, glutathionylspermidinesynthetase; Spd, spermidine; TryS, trypanothione synthetase.5408 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBSby redox cycling drugs such as nifurtimox [10,11]. Inaddition, novel trypanothione-dependent enzymes havebeen identified, such as trypanothione S-transferase[12] and glyoxalase I and II [13–15], that are probablyinvolved in defence against chemical stress. The perti-nence of the effects caused by decreasing trypanothi-one content and thus increased chemical stresshighlight the significance of the biosynthetic enzyme(s)of trypanothione as drug target(s) [16].Trypanothione is synthesized in these medicallyimportant parasites from glutathione (GSH) and sper-midine (Spd) by a monomeric C-N ligase [trypanothi-one synthetase (TryS), EC 6.3.1.9], in a two-stepreaction with glutathionylspermidine as an intermedi-ate [17–20]. Both trypanothione reductase and TryShave been shown to be essential for parasite survival[21–25]. However, in the insect parasite, Crithidiafasciculata, TryS forms a heterodimer with the bi-functional glutathionylspermidine synthetase ⁄ amidase(GspS, EC 6.3.1.8 ⁄ GspA, EC 3.5.1.78) [26]. Previouswork suggested that each biosynthetic enzyme indepen-dently adds successive molecules of GSH to Spd tomake trypanothione [26,27]. However, recombinantTryS from C. fasciculata (CfTryS) has been reportedsubsequently to catalyse both steps of trypanothionesynthesis [28]. Although a gene for GspS has not beenidentified in Trypanosoma brucei, there is a pseudogenein Leishmania major (accession number AJ748279) [19]and putative genes for GspS within the genomes ofLeishmania infantum (accession number AM502243)and Trypanosoma cruzi (accession number EAN98995)that remain to be functionally characterized. Genomesequencing information has also highlighted the pres-ence of GSPS in a range of enteric pathogens such asSalmonella and Shigella [29,30]. The mechanism andphysiological function of this protein are unknown,but in Escherichia coli it is proposed to be involved inregulation of polyamine levels during growth [31], anda similar function has been postulated for C. fasciculataGspS (CfGspS) [32]. Glutathionylspermidine accumu-lates only under stationary-phase conditions, and analternative proposal is that it may be more effective inprotecting DNA from oxidant damage than GSH [33].A previous lack of structural information on thisimportant class of enzymes has been recently resolvedwith the reported crystal structure of GspS fromE. coli (EcGspS), which includes the enzyme in com-plex with substrate, product and inhibitor [34].Preliminary enzyme characterization has previouslybeen described for CfGspS [35], as well as kinetic studieson the partially purified native enzyme using an HPLCmethod [36,37]. Other studies have identified phosphon-ic and phosphinic acid derivatives of GSH as moderateinhibitors of CfGspS [38]. The most active of these wasa phosphonic analogue of GSH (c-l-Glu-l-Leu-GlyP),which displayed linear noncompetitive inhibition(Ki$ 60 lm). This analogue was further improved asan inhibitor of CfGspS by the substitution of the glycinemoiety with amino acid analogues, such as diamino-propionic acid (Ki$ 7 lm) [39]. Although these inhibi-tors are excellent lead compounds for drug designagainst the trypanosomatid parasites, none, as yet, hasyielded Kivalues in the nanomolar range.Proteases that catalyse the direct addition of waterto proteins or peptides proceed via an unstable tetrahe-dral intermediate. These enzymes are inhibited byphosphorus-based stable mimics of the intermediate[40]. Such high-affinity analogues are termed transitionstate analogues or intermediate analogues [41]. Simi-larly, ATP-dependent ligases involve attack of a nucle-ophilic substrate on an electrophilic acyl phosphate[42] via a tetrahedral intermediate. These ligases areinhibited by stable analogues of this intermediate [43–45]. Original work on this type of analogue based onglutathionylspermidine was carried out on EcGspS[46,47]. These studies investigated GSH–Spd conju-gates (Fig. 1), with the objective of developing enzymeinhibitors that block the biosynthesis of trypanothione[46–51]. The synthetase activity of EcGspS was inhib-ited by a phosphonate tetrahedral mimic, in a noncom-petitive, time-independent manner with Ki$ 10 lm[47], and more potently by the phosphinate analoguein a time-dependent manner with Ki*=8nm [46,50].In each case, the phosphorus-based pseudopeptide hadno effect on the amidase activity.Here we examined the kinetic mechanism of CfGspSand determined the modality of inhibition and potencyof these compounds against CfGspS and the homo-logous enzyme TryS from various disease-causingparasites.ResultsInitial velocity analysis of the kinetic mechanismof GspSA matrix of kinetic data was collected in order to deter-mine the kinetic mechanism of GspS. Six families ofkinetic data were generated where each ligand (GSH,Spd and ATP) was treated as the varied substrate atdifferent fixed concentrations of another substrate, main-taining a constant saturating concentration of the thirdsubstrate [52–55]. The corresponding double reciprocalplots of the data are shown in Fig. 2. A ping-pongmechanism can be ruled out, as the fitted lines of theLineweaver–Burk plots converge with each combinationS. L. Oza et al. Kinetics and inhibition of ATP-dependent ligasesFEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5409Fig. 1. Proposed intermediate of glutathionylspermidine and its phosphon(phin)ate analogues.[GSH] = 1000 µM [GSH] = 1000 µM 1/[ATP] (µM–1)0510[Spd] 1000 500 125 250 62.5 [ATP] [Spd] = 1000 µM 1000 500 125 250 62.5 500 125 250 31.25 62.5 [Spd] = 1000 µM [ATP] [ATP] = 500 µM [GSH] 0 0.04 0.08 1/[Spd] (µM–1)0 0.01 0.02 1/[GSH] (µM–1)0 0.01 0.02 1/[ATP] (µM–1)0 0.02 0.04 1/[Spd] (µM–1)0 0.01 0.02 1/[GSH] (µM–1)0 0.01 0.02 1/Rate (s)01231/Rate (s)02461/Rate (s)01231/Rate (s)01231/Rate (s)0121/Rate (s)[Spd] [ATP] = 500 µM 1000 500 125 250 62.5 1000 500 125 250 62.5 500 250 31.25 62.5 125 [GSH] Fig. 2. Kinetic analysis of datasets for GspS. Assay details are described in Experimental procedures. The lines represent the global nonlin-ear fit of the data to the rapid equilibrium random ter-reactant mechanism (Eqn 1) plotted as a Lineweaver–Burk transformation. Reactionrates are reported as catalytic centre activity (s)1).Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.5410 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBSof substrates. After excluding a ping-pong mechanism,the 16 possible models for rapid equilibrium ter-reactant systems were tested, including random,ordered, and hybrid random–ordered [52]. Statisticaltests of each fit revealed that the rapid equilibriumrandom ter-ter model [see Eqn (1), Experimental pro-cedures] fitted significantly better than any other ofthe 15 models (P <10)12). The interaction factorswere close to unity in this model, and when the inter-action factors were set a = b = c = 1, the two fitswere not significantly different (P > 0.05), but didreturn $ 10-fold lower standard errors for the bindingconstants. Thus, the simplest model compatible withthe data suggests that substrates bind to GspS in anyorder, without affecting binding of the other substrates,to form a quaternary complex, enzyme–GSH–ATP–Spd. When a = b = c = 1, the equilibrium dissocia-tion constants for the binding of substrate to the freeenzyme are 609 ± 26, 157 ± 5 and 215 ± 8 lm forGSH, Spd and ATP, respectively, and kcat= 22.8 ±0.6 s)1. When GSH and ATP were varied in a constantratio (10 : 1) versus various concentrations of Spd,they produced a series of Lineweaver–Burk plots thatclearly converged (Fig. 3). This indicates that a productrelease step does not occur between the binding of ATPor GSH and Spd. Thus, the proposed kinetic modelfor GspS is consistent with a random ter-reactantmechanism, as shown in Fig. 4A.Inhibition by phosphonate analogueThe compounds used in this study were designed tomimic the unstable tetrahedral intermediate formedduring GspS-catalysed synthesis of glutathionylspermi-dine (Fig. 1). However, as reported for EcGspS [47],no time-dependent inhibition of CfGspS was observedwith the phosphonate mimic (Fig. 5), which suggeststhat this compound is not acting as a mimic of theunstable intermediate, but as a bisubstrate analogue[56] incorporating key functional groups of both GSHand Spd in the inhibitor. This compound behaves as amodest classical linear competitive inhibitor of GspSwith respect to GSH (Fig. 5B) with a Kiof156 ± 13 lm. Note that for classical reversible inhibi-tors, the rate of product formation is constant pro-vided that there is no significant depletion of substrateor inhibition by product.Inhibition by phosphinate analogueIn contrast to the simple, linear inhibition shown by thephosphonate, time-dependent inhibition was observedfor the phosphinate mimic (Fig. 6A). In reaction mix-tures containing a slow-binding inhibitor initiated bythe addition of enzyme, the initial velocity v0is indepen-dent of inhibitor concentration, but decreases to aslower steady-state velocity vsthat is dependent oninhibitor concentration [41]. These results are consistentwith glutathionylspermidine-dependent phosphoryla-tion of the phosphinate (Fig. 4B), as previously demon-strated for the inhibition of EcGspS [34,46,50]. Theprogress curves for each phosphinate concentrationwere fitted to Eqn (3) (Experimental procedures) toobtain values for v0, vsand kobs. Values for kobswerethen plotted against the inhibitor concentration(Fig. 6B). A linear dependency between [I] and kobswasobserved, and was fitted to Eqn (4) (Experimentalprocedures) to obtain estimates for k3¢ and k4. Theprogress curves used to determine the kobsvalueswere obtained at [S] ⁄ Kmfor GSH of 1.64. The rateconstant k3¢ (2.64 · 104m)1Æs)1) was subsequentlycorrected for competition by substrate, yieldingk3= 6.98 · 104m)1Æs)1(k3= k3¢[1 + [S] ⁄ Km]). They-intercept of Fig. 6B yields an estimate of k4of1.3 · 10)3Æs)1. Thus, the overall dissociation half-lifefor the complex is 0.14 h (enzyme–inhibitor complexhalf-life values were calculated as the ratio of 0.693 ⁄ k4).For an inhibitor of this type, the dissociation constant(Ki) is then obtained from the ratio of k4⁄ k3, yielding aKiof 18.6 nm. To confirm the Kivalue, v0and vsobtained at different concentrations of inhibitor werefitted to the equation vs= v0⁄ (1 + [I] ⁄ Kiapp) by nonlin-ear regression, yielding a Kiappvalue of 31.1 ± 2.1 nm,and true Kivalue was calculated to be 19.0 ± 1.3 nm,using the relationship Ki= Kiapp⁄ (1 + [S] ⁄ Km). Thusboth methods of determining Kiare in excellentagreement.1/[Spd] (mM–1)0 2 4 1/Rate (s)00.40.81.2[GSH:ATP] mM0.5:0.05 0.75:0.075 1:0.1 1.5:0.15 2:0.2 Fig. 3. Lineweaver–Burk analysis of the variation of GSH and ATPat a fixed ratio of 1 : 10 versus Spd concentration. The final con-centrations of GSH and ATP (mM) for each dataset are displayed onthe graph. Reaction rates are reported as catalytic centre activity(s)1).S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligasesFEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5411An alternative approach was used to obtain an inde-pendent estimate of k4. In this method, the enzyme waspreincubated with excess inhibitor ([I] ‡ 10 [E]), andthe reaction was then initiated with substrate. Underthese conditions, a slow release of inhibitor is observeduntil a steady state is reached. Provided that there isno significant enzyme inactivation, substrate depletionor product inhibition, this steady state should be iden-tical to the steady state established when initiating withenzyme [57]. High concentrations of enzyme and inhib-itor were preincubated for 1 h to allow the system toreach equilibrium. Subsequent dilution into a largevolume of assay mix containing saturating substrateconcentrations causes dissociation of the enzyme–inhibitor complex with regain of activity. Under theseconditions, provided that the initial rate v0and theeffective inhibitor concentration are approximatelyequal to zero, the rate of recovery of full enzyme activ-ity will provide k4. When maintaining [I]>[E]([I] = 250 nm,[E] = 20 nm), it proved impossible tomeasure enzyme activity upon 100-fold dilution intothe assay mixture. Instead, high concentrations ofinhibitor (200 lm), enzyme (20 lm) and ATP (400 lm)were preincubated on ice for 60 min and then appliedto a desalting column to remove all free inhibitor. Thefollowing reactions were then analysed: (a) enzyme-only control (Fig. 7, open circles); (b) the completeinhibition reaction, enzyme + inhibitor + ATP (Fig. 7,open squares); and (c) inhibitor-only control added toan equal volume of the enzyme-only control sample(Fig. 7, closed circles). The inhibitor-only controlprogress curve is linear and matches that of theenzyme-only control, demonstrating that essentially noinhibitor has passed through the resin. The regain ofactivity experiment (Fig. 7, open squares) clearly showsthat an enzyme-bound inhibitor complex passesthrough the column and undergoes very slow dissocia-tion upon dilution into the assay mixture. Under theseconditions, both v0and the free inhibitor concentrationshould be negligible in the final assay, so that the rateof recovery of activity provides the value for k4. Afterfitting of the data to Eqn (3) (Experimental pro-cedures), a k4value of 1.36 ± 0.06 · 10)3Æs)1wasobtained, in excellent agreement with the valueobtained previously by varying the concentration ofphosphinate and initiating with enzyme.Modality of inhibitionThe mode of inhibition of the slow-binding phosphi-nate was determined by examining the effect of varyingeach substrate on the value of kobsat a fixed inhibitorconcentration [58]. For a competitive inhibitor, kobsdecreases in a hyperbolic fashion with increasingA B Fig. 4. Model of ter-reactant mechanism ofGspS catalysis and postulated slow-bindinginhibition by the phosphinate mimic. (A)Kinetic mechanism. KGSH, KSpdand KATParethe equilibrium dissociation constants forthe binding of substrate to free GspS (E).(B) Postulated structure of the phosphory-lated phosphinate mimic.Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.5412 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBSconcentrations of substrate. This is observed withGSH or Spd as varied substrate (Fig. 8, closed andopened circles). For a noncompetitive inhibitor, kobsisindependent of substrate concentration (i.e. kobs= k),whereas for an uncompetitive inhibitor, kobsincreasesin a hyperbolic fashion with increasing concentrationsof substrate. As kobsincreases with increasing concen-trations of ATP (Fig. 8, closed squares), this suggestsuncompetitive inhibition. These data were then fittedto the appropriate equation for either competitive inhi-bition [Eqn (5), Experimental procedures] or uncom-petitive inhibition [Eqn (6), Experimental procedures].The respective Kmvalues for GSH, Spd and ATP are400 ± 80 lm, 120 ± 40 lm and 130 ± 26 lm,inreasonable agreement with the respective Kmvaluesdetermined directly in the substrate matrix experimentabove. Thus, the phosphinate inhibitor behaves as aslow-binding competitive bisubstrate inhibitor withrespect to GSH and Spd, but not ATP. The latterobservation is consistent with the hypothesis that anelectrophilic acyl phosphate is formed by reaction ofATP and GSH. The acyl phosphate then reacts withSpd to form an unstable tetrahedral intermediate,which is mimicked by the stable tetrahedral phosphi-nate inhibitor. The nucleotide is not a component ofthe unstable tetrahedral intermediate, and therefore theKi of 156 ± 13 µM[I] = 50 µM0 0.005 0.01 0.015 0246[I] = 100 µM[I] = 200 µM[I] = 400 µM[I] = 0 µMRate (s–1)1/[GSH] (µM–1)0 5 10 15 00.010.020.03AB1000 500 250 100 50 0 Time (min) Product (µmol)Fig. 5. Linear competitive inhibition of GspS by phosphonate ana-logue. (A) Progress curves demonstrating the classical competitiveinhibition of GspS activity by phosphonate. Assays with GspS wereperformed in 250 lL of assay buffer with 10 nM GspS, 1 mM Spd,1mM GSH, 2 mM ATP and various phosphonate concentrations (0–1000 lM) as indicated. The lines fitted to the data points are linearfits for each of the phosphonate concentrations denoted. The linearregression values for all the data points are ‡ 0.997. (B) Kineticanalysis of GspS inhibition by phosphonate. Assays with GspSwere performed in 250 lL of assay buffer with 1 mM Spd, variousGSH concentrations (62.5–2000 lM), various phosphonate concen-trations (50–400 lM) as indicated, and elevated levels of GspS(200 nM). The lines on the Lineweaver–Burk transformation are thebest global nonlinear fit of the data to Eqn (2) describing linear com-petitive inhibition. Reaction rates are reported as catalytic centreactivity (s)1).Time (min)Product (µmol)00.020.040.060.080.1AB010.050.10.250.5[Inhibitor] (µM)0 5 10 150 0.2 0.4 0.6 0.8 1kobs (s–1)00.010.020.03Fig. 6. Slow-binding inhibition of GspS by phosphinate analogue.(A) Assays with GspS were performed as described in Experimen-tal procedures with 15 nM GspS, and various phosphinate concen-trations (0–1 lM) as indicated, with 1 mM each GSH and Spd.(B) Determination of the association rate k3¢ from the plot of kobsas a function of phosphinate concentration. The line represents alinear fit of kobsand [I] values (phosphinate concentrations). Thekobsvalues were calculated from Eqn (4), and the line predicts aslope (k3¢) of 0.026 lM)1s)1.S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligasesFEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5413phosphinate would not be expected to compete withATP in binding to the enzyme.To determine whether the phosphinate is turnedover by CfGspS in the presence of ATP, the activity ofthe enzyme (100 nm) was determined in the absence ofGSH or Spd plus or minus 1 lm phosphinate over30 min. After correction for the background rate dueto auto-oxidation of NADPH and hydrolysis of ATPin the coupling system, the net rates of endogenousATPase activity ($ 0.01% of kcat) in the presence andabsence of inhibitor are 1.4 (± 0.9) · 10)3and3.0 (± 1.5) · 10)3Æs)1, respectively (mean of threedeterminations). This shows that the inhibitor is notturned over by the enzyme. However, this method isinsufficiently sensitive to detect a single phosphoryla-tion event.Inhibition of TryS with phosphinateHaving established that CfGspS is potently inhibitedby the phosphinate inhibitor, it remained to be deter-mined whether the homologous enzyme, TryS, couldalso be inhibited in a similar manner. Owing to thevarious pH optima, Kmvalues for substrates and GSHsubstrate inhibition profiles of the various TrySenzymes to be compared (C. fasciculata, L. major,T. cruzi and T. brucei), a uniform assay was used forIC50determination, i.e. 2 mm Spd, 0.2 mm GSH,2mm ATP, 100 mm (K+) Hepes (pH 7.2). This allowsfor direct comparison of the data collected for all theenzymes under conditions that approximate to thephysiological metabolite levels found in these organ-isms [59]. In this study, IC50values, slope factors andKiappvalues were determined and found to be 20–40-fold less that that of CfGspS (Table 1). In all cases,the slope factor was approximately 1, indicating simplebinding at a single site for all the enzymes tested.DiscussionAn understanding of the kinetic and chemical mecha-nism of GspS and TryS involved in the biosynthesis ofglutathionylspermidine and trypanothione is crucial forthe design of inhibitors against these potential drugtargets. TryS is particularly challenging in this respect,as these enzymes display pronounced high substrateinhibition by GSH and form glutathionylspermidine asan intermediate [17–19]. CfGspS does not displaysubstrate inhibition by GSH [35,60], and thereforeprovides a convenient simple model for this class ofATP-dependent C-N ligases.The kinetic dataset for CfGspS fits best to a rapidequilibrium random ter-ter reaction mechanism, anddefinitively excludes a mechanism where either: (a)ADP is released after phosphorylation of GSH priorto binding of Spd; or (b) ADP is released followingformation of a phosphorylated enzyme intermediate(ping-pong) prior to binding of GSH or Spd. In thisrespect, the mechanism for CfGspS is similar to thatTime (min)0 5 10 15Product (µmol)00.020.040.060.080.1Fig. 7. Rate constant (k4) for dissociation of the GspS–phosphinatecomplex. Three samples were preincubated for 60 min in 100 mMHepes (pH 7.3) on ice. The first contained GspS (20 lM) only, thesecond GspS (20 lM) with excess phosphinate (200 lM) and Mg2+-ATP (400 lM), and the third inhibitor ⁄ Mg2+-ATP only (i.e. noenzyme). All samples were desalted, and the flow-through wasadded to the coupled assay reaction mix in the following combina-tion: flow-through of sample 1 (enzyme-only control, s); flow-through of sample 2 (GspS preincubated with excess phosphinate,h); and flow-through of sample 1 plus sample 3 (i.e. a controlshowing that unbound inhibitor is completely removed using thecolumn method, d), The rate constant associated with the regener-ation of enzymatic activity (k4) was determined as described in thetext.[Varied substrate] (mM)0246810kobs (s–1)00.0050.010.015Fig. 8. Modality of inhibition by phosphinate analogue. The effectof varying GSH (d), Spd (s) and ATP ()onkobswas determinedat a fixed concentration of phosphinate. The hyperbolic fits wereobtained using either Eqn (5) for competitive inhibition (for GSHand Spd) or Eqn (6) for uncompetitive inhibition (for ATP).Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.5414 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBSfor c-glutamylcysteine synthetase from T. brucei [53].However, unlike the case with c -glutamylcysteine syn-thetase, we did not detect any marked influence ofprior binding of one substrate on the equilibrium dis-sociation constants of the other substrates [that is, theinteraction factors a, b and c were all close to unity,and statistical analysis did not favour their inclusion inEqn (1)] (Experimental procedures) [52].Our results are also broadly in agreement with aprevious study which concluded that partially purifiedCfGspS follows a rapid equilibrium random ordermechanism with interaction factors close to unity [37].However, we were unable to reconcile the peptidesequence data reported by Flohe´et al. with our own,as it corresponded to our sequence for CfTryS. Thisdiscrepancy was later corrected in an erratum byFlohe´’s group [36], but raised a second discrepancyconcerning CfTryS. In our hands, heterologous expres-sion of CfTryS did not yield active proteins, whereasFlohe´’s group reported that authentic CfTryS was ableto catalyse the synthesis of trypanothione from GSH,Spd and ATP [28], similar to our findings for TrySfrom T. brucei, L. major and T. cruzi [17–19]. Toresolve this remaining discrepancy, we have repeatedour initial study. The newly cloned enzyme was foundto differ at position 89, with a serine replacing anasparagine in the original construct (AF006615). Thehomogeneously pure soluble protein was found to beactive with either GSH or glutathionylspermidine, andthe product with either substrate was confirmed to betrypanothione by HPLC analysis (data not shown).The reason for our previous failure [27] to detect thisactivity by heterologous expression in yeast is notapparent, but may have been due to a cloning or PCRerror involving this S89N mutation. Nonetheless, wenow agree entirely with the report by Comini et al.[28] that CfTryS is capable of catalysing both steps inthe biosynthesis of trypanothione from GSH and Spd.A kinetic mechanism has not been determined forthe E. coli enzyme, but a reaction mechanism has beenproposed in which the glycine carboxylate of GSH isinitially phosphorylated by the c-phosphate of ATP toform an acyl phosphate, and this is followed by nucleo-philic attack of the N1-primary amine of Spd on theacyl phosphate, leading to the formation of an unsta-ble tetrahedral intermediate [46,48,49]. Structuralstudies on EcGspS in complex with substrates andinhibitors provide strong support for this model[34]. Of particular note was the observation that theslow-binding phosphinate inhibitor [46,50] had beenphosphorylated by ATP to form the tetrahedral phos-phinophosphate in the active site, as previously postu-lated [51]. In addition, a disordered domain in theapoenzyme was observed to adopt an ordered confor-mation over the active site when bound with substratesor inhibitor. Our kinetic studies indicate that all threesubstrates have to bind to the enzyme prior to cataly-sis. This suggests that formation of the quaternarycomplex induces closure of the lid domain over theactive site to form a catalytically competent complex,thereby preventing access of water to hydrolyse theacyl phosphate intermediate.Our kinetic analysis shows that the phosphonateanalogue displays classical, linear competitive inhibi-tion with respect to GSH, with a modest Kiof 156 lmagainst CfGspS, as compared to the mixed-type pat-tern (Kiand Ki¢ of 6 and 14 lm, respectively) reportedfor EcGspsS [47]. In contrast, the phosphinate displaysslow tight-binding inhibition with a Kiof 19 nm, simi-lar to the Ki*of8nm for the E. coli enzyme [46]. Ourstudies also demonstrate that this inhibitor behaves asa mimic of the unstable tetrahedral intermediate that isproposed to form during the GspS-catalysed reactionas originally postulated [51]. At first sight, the uncom-petitive behaviour of the phosphinate inhibitor ratherthan noncompetitive behaviour is not consistent with arapid equilibrium random mechanism. However, suchan inhibition pattern would be expected if the inhibitorunderwent binding followed by a single phosphoryla-tion event, as suggested by the kinetic behaviourTable 1. Inhibition constants of phosphinate against GspS and various TryS enzymes. All assays were performed under conditions approxi-mating to the intracellular physiological state (i.e. pH 7.2, 2 mM Spd, 0.2 mM GSH and 2 mM Mg2+-ATP), and were initiated with 100 nMeach enzyme in the presence of various phosphinate concentrations. IC50values and slope factors are from the inhibition profiles determinedfrom Eqn (7), and Kiappvalues were determined from the tight-binding inhibition equation (Eqn 8). The errors represent the standard error ofthe fit to the appropriate equation.Inhibition constantsEnzymeCfGspS CfTryS L. major TryS T. cruzi TryS T. brucei TrySIC50(nM) 72 ± 6 1380 ± 380 650 ± 25 530 ± 20 1300 ± 50Slope factor 1.2 ± 0.1 1.1 ± 0.3 1.1 ± 0.04 1.1 ± 0.04 1.1 ± 0.05Kiapp(nM) 29 ± 5 1330 ± 350 580 ± 30 490 ± 20 1200 ± 500S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligasesFEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5415observed in this study and others [46,50] and con-firmed in the crystal structure of this inhibitor boundin the active site of EcGspS [34]. The glutathionyl-spermidine phosphinate analogue is also a potentinhibitor of TryS enzymes from L. major, T. cruzi andT. brucei; when assayed under identical conditionsapproximating to intracellular concentrations, TrySenzymes are approximately 20-fold less sensitive thanCfGspS. Although the phosphinate showed no growth-inhibitory activity at 100 lm over 72 h of exposureagainst L. major promastigotes, T. cruzi epimastigotesand T. brucei procyclics, various chemical modifica-tions could enhance cellular penetration, e.g. acyloxyester prodrugs [61].An alignment of EcGspS with CfGspS and otherTryS proteins reveals some other interesting features(Fig. 9). First, despite the trypanosomatid proteinshaving < 30% identity and < 45% similarity, allthree residues involved in binding Mg2+(green trian-gles) and three of four involved in binding ATP (redtriangles) are absolutely conserved. Second, four of fiveresidues interacting with GSH (blue triangles) in theproductive binding mode are also conserved. Third,two of three residues implicated in binding of the Spdmoiety of the phosphinate inhibitor (yellow triangles)are also conserved. Fourth, Pai et al. also noted a non-productive binding mode (black triangles), where GSHforms a mixed disulfide with Cys338 and an isopeptidebond between the glycine moiety of GSH and Lys607of the protein. However, this is clearly not required forcatalysis in the trypanosomatid enzymes, as neitherresidue is conserved in any of these enzymes. Finally,the E. coli enzyme is a homodimer, whereas the try-panosomatid TryS enzymes are monomeric, or hetero-dimeric in the case of Cf TryS and CfGspS. In thiscase, the residues that interact between monomers inEcGspS (black circles) are hardly conserved at all. Oneother interesting difference between EcGspS andCfGspS is that the latter enzyme has an additional 100amino acids. The alignment in Fig. 9 highlights a num-ber of insertions that are dispersed throughout thesequence of CfGspS. These include an insertion of 17amino acids in the amidase domain and two in thesynthetase domain (one of 14 amino acids and theFig. 9. Conservation of key functional residues identified for EcGspS in CfGspS and TryS. The GenBank ⁄ EMBL ⁄ DDBJ accession numbersused to generate the alignment usingT-COFFEE are: EcGspS (U23148), CfGspS (U66520), CfTryS (AF006615), L. major TryS (AJ311570),T. cruzi TryS (AF311782) and T. brucei TryS (AJ347018). Absolutely conserved residues are marked in bold; coloured residues indicate sidechain interactions in EcGspS with substrates or inhibitors [33]. Green triangles, residues involved in binding Mg2+; red triangles, three of fourresidues involved in binding ATP; blue triangles, four of five residues interacting with GSH; yellow triangles, two of three residues implicatedin binding of the Spd moiety of the phosphinate inhibitor; black triangles, nonproductive binding mode, where GSH forms a mixed disulfidewith Cys338 and an isopeptide bond between the glycine moiety of GSH and Lys607 of the protein; black circles, residues that interactbetween monomers in EcGspS. Only the relevant C-terminal region of the synthetase domain is shown.Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.5416 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBSother of 39 amino acids). It may be that these addi-tional insertions in CfGspS are required for its hetero-dimeric interactions with CfTryS.From the above analysis, it is not immediately obvi-ous why the phosphinate inhibitor is $ 20-fold lesspotent against the TryS enzymes than against CfGspSand EcGspS. Possibly, the substitution of Asp610,which is involved in recognition of the N8-amine ofSpd, for a proline in TryS (methionine in CfGspS) is acritical factor. Alternatively, the fact that TryS has toaccommodate either N1-glutathionylspermidine orN8-glutathionylspermidine as well as Spd in the poly-amine-binding site may be a significant factor. Thecurrent ligand-free structure of L. major TryS [62] isnot helpful in resolving these issues, and substrates orinhibitors in complex with TryS are needed. In themeantime, the phosphinate inhibitors represent a valu-able starting point for further development of drug-likeinhibitors against this target.Experimental proceduresMaterialsAll chemicals were of the highest grade available fromSigma-Aldrich (Gillingham, UK), Roche Diagnostics Ltd(Burgess Hill, UK) or Calbiochem (Merck Biosciences,Nottingham, UK). The phosphonate and phosphinateanalogues of glutathionylspermidine were synthesized aspreviously described [49,51]. The structure and purity ofboth compounds were confirmed by NMR, high-resolutionMS and elemental analysis.Expression and purification of GspSRecombinant GspS was prepared using a 60 L fermenter,and purified to greater than 98% homogeneity as describedpreviously [35], except that a HiLoad Q Sepharose 16 ⁄ 10column (GE Healthcare, Amersham, UK) was used in thefinal step. Active fractions were pooled, buffer wasexchanged into 100 mm (K+) Hepes containing 0.01%sodium azide, 1 mm dithiothreitol and 1 mm EDTA, and thesample concentration was determined using the calculatedextinction coefficient of 99 370 at 280 nm. Aliquots of GspSwere then flash frozen and stored in aliquots at )80 °C.Expression and purification of TryS enzymesTryS enzymes from T. brucei, L. major and T. cruzi wereprepared as described previously [17–19]. In addition, wewere able to obtain functionally active CfTryS by generat-ing a new construct in a modified pET15b vector in whichthe thrombin cleavage site had been replaced by a TEVprotease cleavage site. The ORF was PCR amplified fromC. fasciculata genomic DNA using the sense primer 5¢-CAT ATG GCG TCC GCT GAG CGT GTG CCG G-3¢,which includes an NdeI site (underlined) and a start codon(in bold), and the antisense primer 5¢-GGA TCC TTA CTCATC CTC GGC GAG CTT G-3¢, which includes a stopcodon (in bold) and a BamHI site (underlined); the PCRproduct was subsequently cloned, via pCR-Blunt II-TOPO(Invitrogen, Paisley, UK), into the NdeI ⁄ BamHI site ofpET15bTEV. Sequencing of three independent clonesrevealed that the sequence was almost identical to thesequence previously deposited for CfTryS (AF006615),except that serine replaced asparagine at position 89 of theORF. This construct, CfTryS_pET15bTEV, was trans-formed into BL21(DE3)pLysS-competent cells (Novagen,Merck Biosciences); typically, cultures were then grown inTerrific Broth at 37 °CtoD600 nm‡ 1.2, cooled to 22 °C,induced with a final concentration of 0.5 mm isopropyl-b-d-thiogalactoside, and grown for an additional 16 h.Purification of recombinant protein was achieved using twochromatographic steps [5 mL His-Trap (GE Healthcare),TEV protease cleavage (2 h, 30 °C), followed by a HiLoadQ Sepharose 16 ⁄ 10 HP column (GE Healthcare)].Assay conditions for the kinetic mechanismof GspSAll kinetic assays were performed at 25 °C using an assaysystem that couples ADP production to NADH oxidation at340 nm [35]. Each assay contained 100 m m (K+) Hepes(pH 7.3), 0.2 mm NADH, 1 mm phosphoenolpyruvate,5mm dithiothreitol or Tris(2-carboxyethyl)phosphine hydro-chloride, 0.5 mm EDTA, 10 mm MgSO4,2UÆmL)1l-lactatedehydrogenase, and 2 UÆmL)1pyruvate kinase (both cou-pling enzymes were from rabbit muscle, and purchased fromRoche), with varying amounts of ATP, GSH and Spd in atotal volume of 1 mL. Rates are expressed in moles of sub-strate utilized per second per mole of enzyme. To determinethe kinetic mechanism, data were collected for GspS at arange of substrate concentrations. A complete matrix ofrates as a function of substrate concentration (ATP, 31.25–500 lm; GSH, 62.5–1000 lm; and Spd 62.5–1000 lm) wasgathered, so that for any given concentration of any one sub-strate the rates were measured over the entire range of theremaining two substrates. When fixed concentrations of eachof these substrates were used, the final concentrations forATP, GSH and Spd were 0.5, 1 and 1 mm respectively,unless otherwise stated. The assay was initiated by addingGspS (300 nm) and, after a lag of 10 s, the linear decrease inabsorbance was monitored for up to 1 min. Data were thenglobally fitted by nonlinear regression to all possible modelsfor rapid equilibrium ter-reactant systems [52]. The goodnessof fit for each model was compared statistically using theF-test and kinetic constants obtained by fitting to Eqn (1):S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligasesFEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5417[...]... 279, 4272 6–4 2731 Kinetics and inhibition of ATP-dependent ligases 56 Broom AD (1989) Rational design of enzyme-inhibitors multisubstrate analog inhibitors J Med Chem 32, 2–7 57 Morrison JF (1982) The slow-binding and slow, tightbinding inhibition of enzyme-catalyzed reactions Trends Biochem Sci 7, 10 2–1 05 58 Copeland RA (2005) Tight binding inhibition In Evaluation of Enzyme Inhbitors in Drug Discovery:... constants for the binding of substrate with free enzyme, and a, b and c are the interaction factors between Spd and ATP, GSH and ATP, and GSH and Spd, respectively Inhibitors and enzyme inhibition assays Inhibitor studies employed the coupled assay described above Possible inhibition of the coupling enzyme system was excluded by substituting glucose and hexokinase for GspS or TryS in the assays, in which case... analog of glutathionyl spermidine (GSP), a potent, slow-binding inhibitor of GSP synthetase Bioorg Med Chem Lett 7, 50 5–5 10 Chen SJ & Coward JK (1998) Investigations on new strategies for the facile synthesis of polyfunctionalized phosphinates: phosphinopeptide analogues of glutathionylspermidine J Org Chem 63, 50 2–5 09 Segel IH (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state... phosphinate (200 lm) and Mg2+-ATP (400 lm), in 30 lL of assay buffer at 4 °C for 1 h, in order to reach equilibrium A sample containing only inhibitor and Mg2+-ATP sample was also included as an internal control to verify efficient retention of the phosphinate by the column Following preincubation, samples 5418 k 1 þ ð½SŠ=Km Þ ð5Þ or uncompetitive inhibition (Eqn 6) i Àkt were applied to 0.5 mL of Zeba... analogs of glutathione and glutathionylspermidine: potent, selective binding inhibitors of the E coli bifunctional glutathionylspermidine synthetase ⁄ amidase Chem Biol 4, 85 9–8 66 Kwon DS, Lin CH, Chen SJ, Coward JK, Walsh CT, Bollinger JM Jr (1997) Dissection of glutathionylspermidine synthetase ⁄ amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains J... equation assumes that y falls with increasing [I] The Kiapp values of FEBS Journal 275 (2008) 540 8–5 421 ª 2008 The Authors Journal compilation ª 2008 FEBS S L Oza et al Kinetics and inhibition of ATP-dependent ligases the inhibitor against each enzyme were determined using the following tight-binding inhibition equation [41] (Eqn 8), where the enzyme concentration [E] was fixed at 100 nm: app ð½EŠ þ... brucei c-glutamylcysteine synthetase characterization of the kinetic mechanism and the role of Cys-319 in cystamine inactivation J Biol Chem 273, 2631 7–2 6322 Jez JM, Cahoon RE & Chen SX (2004) Arabidopsis thaliana glutamate-cysteine ligase functional properties, kinetic mechanism, and regulation of activity J Biol Chem 279, 3346 3–3 3470 Jez JM & Cahoon RE (2004) Kinetic mechanism of glutathione synthetase... phosphinic acid mimic of the tetrahedral reaction intermediate Biochem Pharmacol 65, 31 5–3 18 Bartley DM & Coward JK (2005) A stereoselective synthesis of phosphinic acid phosphapeptides corresponding to glutamyl-gamma-glutamate and incorporation into potent inhibitors of folylpoly-gamma-glutamyl synthetase J Org Chem 70, 675 7–6 774 Lin C-H, Chen S, Kwon DS, Coward JK & Walsh CT (1997) Aldehyde and phosphinate... Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress Mol Microbiol 35, 54 2–5 52 FEBS Journal 275 (2008) 540 8–5 421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5419 Kinetics and inhibition of ATP-dependent ligases S L Oza et al 24 Comini MA, Guerrero SA, Haile S, Menge U, Luns´ dorf H & Flohe L (2004) Validation of Trypanosoma brucei trypanothione. .. J 274, 564 3–5 658 6 Schlecker T, Comini MA, Melchers J, Ruppert T & Krauth-Siegel RL (2007) Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei Biochem J 405, 44 5–4 54 7 Fairlamb AH & Henderson GB (1987) Metabolism of trypanothione and glutathionylspermidine in trypanosomatids In Host–Parasite Cellular and Molecular Interactions in Protozoal Infections . ATP-dependent ligases in trypanothione biosynthesis – kinetics of catalysis and inhibition by phosphinic acid pseudopeptides Sandra L. Oza1,. valueobtained previously by varying the concentration of phosphinate and initiating with enzyme.Modality of inhibition The mode of inhibition of the slow-binding
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