Trypanothione reductase from Leishmania donovani Purification, characterisation and inhibition by trivalent antimonials

Transcription

1 Eur. J. Biocem. 230, (1995) 0 FEBS 1995 Trypanotione reductase from Leismania donovani Purification, caracterisation and inibition by trivalent antimonials Mark L. CUNNINGHAM and Alan H. FAIRLAMB Biocemistry and Cemoterapy Unit, Department of Medical Parasitology, London Scool of Hygiene and Tropical Medicine, England (Received 27 February 1995) EJB /4 Trypanotione reductase was purified to omogeneity from Leismania donovani promastigotes transfected wit te expression plasmid ptexldtr. Te pysical, spectral and kinetic properties were found to be similar to tose obtained from oter patogenic trypanosomatids. Te substrates trypanotione disulfide and exibit MicaelisMenten saturation kinetics wit K,,, values of 36 pm and 9 pm, respectively, te former yielding a k,,,lk, of 5.0X lo6 M s?. Like oter trypanotione reductases, te leismania enzyme is unable to use glutatione disulfide as substrate. Bot trypanotione reductase and te analogous mammalian enzyme, glutatione reductase, are inibited by trivalent but not pentavalent antileismania1 antimonials. Inibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a timedependent manner, wit te pseudofirstorder rate constants of inibition being linearly related to drug concentration. Inibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzymedrug complex is reaced. MelT, an adduct of melarsen oxide and diydrotrypanotione wic is a competitive inibitor of te disulfide binding site of trypanotione reductase, confers protection against Triostam. Prior reduction of te catalytically active disulfide bridge by is essential for inibition. Spectral analysis sows tat te broad absorbance band centred on 530 nm, caracteristic of te cargetransfer complex in te twoelectronreduced EH, enzyme, is lost upon addition of Triostam. Furter spectral canges resemble tose associated wit reduction of te FAD prostetic group to FADH,. Inibition by Triostam is readily reversed by dilution or addition of te ditiols 2,3captopropanol, 2,3captosuccinate or ditiotreitol, but not diydrotrypanotione, suggesting tat tis trypanosomatidunique metabolite is unlikely to protect te enzyme from inibition in wole cells. A mecanism consistent wit tese observations is proposed. Keywords. Trypanotione reductase; glutatione reductase; antimonials ; Leismania donovani. Species of te parasitic protozoa Leismania are estimated to treaten some 350 million people worldwide wit a broad range of diseases. Arguably te most serious of tese diseases is visceral leismaniasis, caused by Leismania donovani and L. infanturn, wic is invariably fatal if untreated. Pentavalent antimonials suc as sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime) are currently te firstcoice drugs for te treatment of visceral leismaniasis [l, 21, despite teir toxicity [3] and te emergence of parasite resistance [4]. Moreover, despite extensive investigation [5 81, te molecular basis for te selective antileismania1 action of tese drugs remains unknown, altoug it is generally considered tat antimonials are only active following bioreduction to te trivalent SbIII form [9]. Given te limitations of te current treatments for leismaniasis, it is clear tat new cemoterapeutic agents are required. Correspondence to A. H. Fairlamb, Biocemistry and Cemoterapy Unit, Department of Medical Parasitology, London Scool of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, England Abbreviations. MelT, adduct of melarsen oxide and diydrotrypanotione ; Pentostam, pentavalent sodium antimonyl gluconate; Triostam, trivalent sodium antimonyl gluconate ; stibocaptate, exasodium 2,2 [ (1,2dicarboxy 1,2etanediyl)bis(tio)] bis 1,3,2ditiastibolane 4,5dicarboxylate. Enzymes. Trypanotione reductase (EC ) ; glutatione reductase (EC ). A rational approac to te design of new antileismanials as identified a number of aspects in leismania biocemistry wic may be exploited. One suc target is te intracellular ditiol trypanotione [W,iVbis(glutationyl)spermidine] [lo], unique to all kinetoplastida including Leismania spp and te parasites responsible for Cagas disease (Trypanosoma cruzi) and African trypanosomiasis (Trypanosoma brucei spp). Trypanotione (or omotrypanotione in T cruzi [ll]) is te principal lowmolecularmass tiol in tese parasites and appears to ave subsumed many of te antioxidant and oter protective functions ascribed to glutatione in oter organisms including mammals (see review [12]). Te intracellular level of diydrotrypanotione and ence te reducing environment is maintained by te dependent flavoproteindisulfideoxidoreductase trypanotione reductase. Altoug structurally and mecanistically analogous to te mammalian enzyme glutatione reductase, trypanotione reductase is sufficiently different in its disulfidebinding site for selective cemoterapeutic attack [ To date, trypanotione reductase as been purified to omogeneity from I: cruzi [21], T congotense (using eterologous expression in Escericia coli) [22] and te insect parasite Critidia fasciculata [23] ; owever, te enzyme as not yet been isolated from any species of Leismania. We ave recently reported tat te trypanolytic trivalent arsenical melarsen oxide is a potent inibitor of trypanotione reductase in vitro, via covalent modification of te vicinal redox

2 Cunningam and Fairlamb (Em J. Biocern. 230) 46 1 active cysteine residues witin te disulfidebinding site [24]. However, witin te intact parasite, diydrotrypanotione appears to protect tese protein tiols by sequestering te arsenical as te stable adduct MelT, itself a competitive inibitor of trypanotione reductase [25]. Altoug uman glutatione reductase is also inibited by melarsen oxide, te parasite enzyme is considerably more sensitive tan its mammalian counterpart [24]. Te similarity in cemical properties of trivalent arsenicals and antimonials prompted us to examine weter tese latter compounds also inibit tese enzymes. Here we report te results of a comparative study on te susceptibilities of L. donovani trypanotione reductase and uman glutatione reductase to antileismanial antimonials. MATERIALS AND METHODS Materials. Cromatograpy resins and oter reagents were obtained as previously [24]. Homogeneous uman erytrocyte glutatione reductase, purified as publised [26], was kindly provide by Dr K. Smit. Triostam (trivalent sodium antimonyl gluconate) and pentostam (pentavalent sodium antimonyl gluconate) from Wellcome Researc Laboratories (Beckenam, Kent) were generously provided by Dr Simon Croft. Geneticin (G418) was purcased from Gibco. All oter reagents and cemicals were of analytical grade. Cultivation of Leismania donovani promastigotes. L. donovani promastigotes (strain LV9, MHOM/ET/67/HU3) were cultivated wit saking in 21 conical flasks at 22 C reacing typical cell densities of 4X107/ml. Te medium consisted of a modified minimum essential Eagle medium containing ribonucleotides and deoxyribonucleotides supplemented wit, 3 gfl glucose,lo g/l Hepes, 2.5 g/l sodium bicarbonate, 0.58 g/l Lglutamate, 5 mgfl folic acid, 2 mgfl biotin, 5 mgfl emin and 40 mga adenine. Medium was titrated to ph 7.5 wit 1 M NaOH prior to filter sterilisation. For cultivation of strains arbouring ptex LdTR [27], medium also contained 100 pg/ml geneticin. Immediately prior to use, 10% eatinactivated fetal calf serum, 87 unitslml penicillin and 87 pg/ml streptomycin were added. Cells were arvested by centrifugation in a fixedangle rotor (6000 g, 30 min, MSE Hig speed 18) and pellets stored at 20 C. Enzyme assays. Trypanotione reductase from L. donovani was assayed spectropotometrically following te trypanotionedependent oxidation of at 340 nm at 27 C. Te standard assay system (0.5 ml) consisted of 20mM Hepes ph 7.4,30 mm NaCl, 0.1 mm EDTA, 150 pm and te reaction was initiated by te addition of 50 pm trypanotione disulfide. One unit of activity (U) is defined as te amount of enzyme required to catalyze te conversion of 1 pmol to NADP+/min at 27 C. Canges in absorbance were monitored wit a Beckman DU70 temperatureregulated spectropotometer. Glutatione reductase was assayed under identical conditions except tat te reaction was initiated by te addition of 100 pm glutatione disulfide. Determination of optimum assay conditions. Ionic strengt and ph optima were determined using a preliminary batc of L. donovani trypanotione reductase wic was approximately 90% pure as judged by SDSPAGE (specific activity = 92 U mgi). Te ionic strengt/activity profile was obtained by addition of increasing amounts of NaCl to 20 mm Hepes ph 7.8 in te presence of 150 pm, te reaction being initiated by addition of 50 pm trypanotione disulfide. Eac concentration of NaCl was assayed in triplicate. Te pwactivity profile over te range was obtained using 20 mm Mes, Hepes or Ces buffers (ph , and respectively). For eac ph, te ionic strengt due to ionised buffer was calculated and NaCl added suc tat te final ionic strengt was optimal, ie. 40 mm. Eac assay consisted of 20 mm buffer at te relevant ph, NaCl as required and 150pM, te reaction being initiated by 50 pm trypanotione disulfide and eac ph assayed in triplicate. Protein concentration. Protein concentrations were determined by te Bradford metod [28] (BioRad) using bovine serum albumin as standard. Te concentration of omogeneous trypanotione reductase was determined using an absorption coefficient of 11.5 mm cm at 463 nm for te oxidized enzyme and a subunit mass of Da derived from te gene sequence WI. Trypanotione reductase purification. Buffers. Trypanotione reductase was purified to omogeneity from L. donovani overexpressing te enzyme following transformation wit plasmid ptexldtr [27]. Te buffers used in te purification were : buffer A, 20 mm potassium diydrogen pospate adjusted to ph 7.2 wit 1 M KOH, 1 mm EDTA; buffer B, 20 mm Bistrispropane ph 7.4, 1 mm EDTA. Bot buffers were supplemented wit 1 mm ditiotreitol immediately prior to use and all manipulations were performed at 4 C. Extraction. L. donovani promastigotes, (9.2 g packed cells, previously frozen and tawed tree times to abolis infectivity) were resuspended in 100 ml buffer A supplemented wit 5 mm benzamidine, 5 mm penantroline and 100 pm penylmetylsulfonyl fluoride. Microscopic examination revealed no intact cells at tis point. Te resulting extract was sonicated (five 30s pulses wit intermittent cooling on ice) followed by centrifugation to remove cellular debris ( g, 30 min). Te supernatant was adjusted to 0.4 % (massfvol.) wit protamine sulfate and gently stirred for 20 min. Precipitated nucleic acids were pelleted by centrifugation (15000 g, 30 min) and te supernatant dialysed extensively against buffer A (fraction I). Afinity cromatograpy using 2 :5 ADPSeparose. Fraction I was applied to a column (0.8 cmxll cm) of adenosine 2,5 bispospate linked to Separose (2,5 ADPSeparose) previously equilibrated wit buffer A. Te column was wased extensively wit buffer A (35 column volumes) after wic a broad yellow band at te top of te resin was observed. Trypanotione reductase was eluted wit 8 mm NADP in buffer A and active fractions (corresponding to te yellow band) collected. Tese fractions were pooled and dialysed against buffer B (fraction 11). Anionexcange cromatograpy using QSeparose. Fraction I1 was applied to a QSeparose anionexcange column (0.8 cmxl3 cm) previously equilibrated wit buffer B, a yellow band forming at te top of te resin. Te column was wased torougly wit buffer B (18 column volumes) and trypanotione reductase subsequently eluted wit a linear salt gradient (0 0.4 M KCl in buffer B). Fractions were collected and tose containing trypanotione reductase activity pooled. Te pooled sample was dialysed against buffer B containing 0.15 M KCl and subsequently concentrated to 1.8 ml witout loss of activity using an Amicon P30 concentrator (fraction 111). Gel filtration cromatograpy (FPLC). Fraction 111 was applied to a gelsizing column (Parmacia Hiload 16/60 Sepadex 200) previously equilibrated wit buffer B plus 0.15 M KCl. A flow rate of 1 ml/min was employed and 2ml fractions collected. Aliquots of fractions containing trypanotione reductase activity were found by SDSPAGE gel to contain a minor contaminant of approximately 100 ma. Te purest fractions were pooled (fraction IV), concentrated to 1.1 ml using an Amicon P 30 concentrator and reapplied to te gelsizing column using a flow rate of 0.75 mymin and collecting 1ml fractions. As above, active fractions were analysed by SDSPAGE, confirming tat te minor contaminant ad been removed. Homogenous

3 462 Cunningam and Fairlamb (Eul: J. Biocern. 230) Table 1. Purification of trypanotione reductase from Leismania donovuni transfected wit ptexldtr. Step Fraction Volume Activity Total Protein Total Specific Yield activity concn protein activity ml U/ml U mg/ml mg U/mg % Cellfree extract I ,5 ADPSeparose I QSeparose Gel filtration 1 rv Gel filtration 2 V fractions were pooled (fraction V) and stored in small aliquots at 20 C in 50% glycerol. Determination of subunit mass. Subunit mass was determined by SDSPAGE wit te following molecular mass standards: myosin, 200 kda; /Igalactosidase, kda; posporylase b, 97.4 kda; bovine serum albumin, 66 kda; ovalbumin, 45 kda; carbonic anydrase, 31 kda; trypsin inibitor, 21.5 kda; lysozyme 14.4 kda. Native molecular mass was determined using a gelsizing column (Parmacia HiLoad 16/60 Sepadex 200) wit te following molecular mass standards : blue dextran, void volume; /Iamylase, 200 kda; alcool deydrogenase, 150 kda; 2 cruzi trypanotione reductase, 108 kda; bovine serum albumin, 66 kda; superoxide dismutase, 32.6 kda; carbonic anydrase, 29 kda; trypsin, 24 kda; insulin, 5.75 kda. Spectra. All spectra were collected on a Beckman DU70 temperature regulated spectropotometer using 1 cm patlengt quartz microcuvettes (100 pl minimum working volume). Data were acquired over a range of nm at a rate of 600 nm/ min. Enzymes were extensively dialysed against assay buffer prior to use. Absorption coefficient of oxidized trypanotione reductase. Te enzymebound flavin was liberated by termal denaturation at 100 C for 20 min in te presence of 10 mm MgC1,. Denatured protein was removed by microcentrifugation and te concentration of free flavin determined from its absorption coefficient at 450 nm (11.3 mm cmi). Enzyme inibition. All incubations were at room temperature and ten warmed to 27 C for 1 min immediately prior to enzyme assay. Addition of drug corresponds to time = 0. Enzymic activities were monitored by removing 475pl aliquots and assaying by te addition of substrate to a final volume of 500 pl. All activities were expressed as a percentage of te control time zero value. Kinetic data was fitted using te nonlinear regression data analysis programme ENZFITTER (ElsevierlBiosoft) written by Dr R. J. Leaterbarrow. HPLC analysis. Trypanotione, antimonial and arsenical compounds were separated by ionpaired reversepase HPLC using a Beckman Ultraspere C18 column and detected by fluorescence following postcolumn derivatisation wit fluorescamine, as publised [30]. RESULTS Purification of Leismania donovani trypanotione reductase. Trypanotione reductase was purified to omogeneity from L. donovani transfected wit te expression plasmid ptex LdTR wit an overall yield of 26% and specific activity of U/mg (see Table 1). A specific activity of 2.1 U/mg in te crude extract represents a 10fold increase in trypanotione reductase levels relative to untransformed cells, 0.2 U/mg, consistent wit publised values [27]. Following column croma tograpy on 2,5 ADP Separose, QSeparose and te first gel filtration step, SDSPAGE revealed a single minor contaminant of subunit mass 32 kda. Tis contaminant was removed by a second gel filtration step using a lower flow rate, fraction V displaying a symmetrical A,,, nm elution profile from te column and a single band on SDSPAGE. Using a similar protocol to tat above, purification of trypanotione reductase from 36 g (packed cells) of untransformed L. donovani promastigotes yielded comparable specific activities at steps I1 and 111, 8.3 and 77.5 U/mg, respectively. However, prior to step IV, ammonium sulfate precipitation (70% saturation) was used to concentrate te sample as opposed to ultrafiltration. Tis resulted in a decrease in specific activity of approximately 50 % (77.5 to 40.6 U/mg) wic proved irreversible despite extensive dialysis against eiter assay buffer or assay buffer supplemented wit 5 mm EDTA. Subsequent gel filtration of tis sample gave a protein wic exibited a single band on SDSPAGE wit a specific activity of 55.1 U/mg. Apart from te lower k,, (5600 mini), te K, values for trypanotione disulfide and and te oter pysical properties were indistinguisable from tose described below for trypanotione reductase purified from transformed cells. Te reason for tis loss in activity is not known. Pysical and cemical caracterisation. A native molecular mass of 106 t 1 kda (n = 3) was obtained from gel filtration cromatograpy. A subunit mass of 50.9 & 1.2 kda (n = 3) was obtained using SDSPAGE, in reasonable agreement wit te value calculated from te publised amino acid sequence, Da [29], and consistent wit a ic structure, as wit oter trypanotione reductases and glutatione reductases (see Table 2). Trypanotione reductase activity was found to be igly dependent upon ionic strengt, te optimum corresponding to 40mM, similar to tat reported for te 2 cruzi enzyme [31]. Increasing ionic strengt results in greatly decreased activity, < 5% of te optimum remaining after addition of 1 M NaCl (data not sown). Under conditions of constant concentrations of trypanotione disulfide and and of ionic strengt equal to 40 mm, te enzyme displayed a broad ph optimum over te range wit maximum activity at 7.4. Outside tis range, activity decreased, wit discernable soulders at ph 8.2 and to a lesser extent at ph 6.6 (data not sown). Trypanotione reductase from L. donovani exibited ig specificity for trypanotione disulfide and as substrates, bot of wic displayed MicaelisMenten saturation kinetics (data not sown). Te K,,, for trypanotione disulfide wit saturating was 36 (? 3) pm wile te apparent K, for wit 150 pm trypanotione disulfide was 9.0 ( 5 1.6) pm. As wit oter trypanotione reductases, glutatione disulfide was a poor substrate, te reduction of 50 mm glutatione disulfide being less tan 0.02 % of tat obtained wit 50 pm trypanotione disulfide. Te enzyme was igly specific for

4 Cunningam and Fairlamb (Eur. J. Biocern. 230) 463 Table 2. Pysical and kinetic parameters of L. donovuni trypanotione reductase. Te subunit masses were taken from te following references : L. donovuni [29], T cruzi [39], Z congolense [40], C. fusciculuta 1411; oter data were taken from: 1: cruzi [21], Z congolense [22], C. fusciculutu [23] (K, for MelT [25]), uman erytrocytes [38]; n.d., not determined. Parameter Trypanotione reductase L. donovani Z cruzi Glutatione reductase uman Z congolense C. fasciculutu erytrocytes Subunits mass (ma) calculated from: amino acid sequence SDSPAGE Oligomeric structure Pyridine dinucleotide Flavin n.d FAD 53.4 FAD 54 FAD 52.5 FAD Absorbance (oxidized) : A, (nm) E,, at A,,,, (M' cm') ? Carge transfer in reduced enzyme eo at A,,,, (mm' cm') 4.2 t Km (pm) trypanotione dilsufide N'glutationylspermidine disulfide glutatione disulfide ? k,,, (min') trypanotione disulfide glutatione disulfide k,,/k, (M' s') K, for MelT (pm) <2 5.0 x lo X 10' X lo X lo X 106 as electron donor, te enzymic rate using 150 pm NADH being only 1.0 ( 5 0.1)% of tat obtained wit 150 pm. Te k,,, for trypanotione disulfide at saturating was min', yielding a k,,,/k, of 5.OX1O6 M' sp, in good agreement wit values obtained for T cruzi and i? congolense enzymes, toug tese values were 2 3fold lower tan tose for C. fasciculata trypanotione reductase (see Table 2). Te enzyme is reasonably stable on storage at 20 C in 50% (by vol.) glycerol, wit a loss of 20% activity over 12 monts. Spectroscopic caracterisation. L. donovani trypanotione reductase possesses spectral properties closely resembling tose of oter trypanotione reductases and glutatione reductases (Table 2 and Fig. 1). Te oxidized enzyme (Fig. 1, trace A), were te redoxactive cysteine residues, Cys52 and Cys57, witin te disulfide binding site are covalently linked as a disulfide bridge, exibits maxima at 377 and 463 nm. Termal liberation of te flavin prostetic group yields an absorption coefficient at 463 nm of 11.5 mm' cm' (standard deviation = 0.2 mm' cm', n = 5). Addition of 2 mm as reductant (Fig. 1, trace B), leads to a decrease in te absorbance at 463 nm wit concomitant acquisition of a broad longwavelengt absorption band centred on 530 nm due to reduction of te cysteine disulfide bridge and formation of a caracteristic cargetransfer complex between te FAD and te proximal sulfydryl group. Tis spectrum is stable indefinitely in te presence of excess reductant. Sitedirected mutagenesis studies on te 7: cruzi enzyme as identified te cargetransfer cysteine as tat nearer te Cterminal, namely C57 in tis case [32]. Inibition of trypanotione reductase by antimonial compounds. A variety of antimonial compounds, in bot te trivalent and pentavalent oxidation states, were examined for teir 12 I I, I ED SO aoa Wavelengt (nm) Fig. 1. Visible absorption spectra of L. donovuni trypanotione reductase. Trace A, oxidized enzyme; trace B, reduced enzyme, 20 min after addition of excess (2 mm); traces CF were obtained at 1, 3, 6 and 10 min, respectively, after addition of 50 pm Triostam to 5.6 pm enzyme after prior reduction wit 2 mm. After 40 min, a furter aliquot of Triostam was added to a final concentration of 100 pm and trace G was obtained 10 min subsequently. Te inset sows te timedependent cange in absorbance at 530 nm (0) and 463 nm (0) after te addition of Triostam to reduced enzyme. ability to inibit L. donovani trypanotione reductase (Table 3). As wit our previously publised work on C. fasciculata trypanotione reductase [24], incubation wit alone causes considerable inibition over 60 min (31 %), wilst in te absence of tis cofactor enzymic activity remains completely stable. Te presence of te trivalent antimonial Triostam results in significantly iger inibition relative to te appropriate con

5 464 Cunningam and Fairlamb (Eul: J. Biocem. 230) Table 3. Inibition of trypanotione reductase by antimonial compounds. Trypanotione reductase (3.7 nm) was incubated, were required, wit (150 pm) for 10 rnin after wic te test compound was added and te samples incubated at 25 "C for 60 min, te final concentration of antimony being constant at 20 pm. Relative activity is expressed as a percentage of te activity remaining after 60 min relative to te activity at zero time. Relative inibition is calculated wit respect to te relevant control activity at 60 min. Results are means 2 standard deviations for triplicate assays. Significance was determined by paired student's ttest, wit an asterisk (*) indicating P< 0,001. A Additions Relative Relative activity inibition % None" Triostam", 20 pm (SbIII) + Triostam, 20 pm (SbIII) + Pentostam, 20 pm (SbV) + potassium antimony tartrate, 20 pm (SbIII) + stibocaptate, 10 pm (SbIII) + antimony triclorideb, 20 pm (SbIII) i i ? i ? * 76.1 * * * a added 30 s prior to trypanotione disulfide. Contains 10% (by vol.) metanol. trol, altoug te inibition observed wen is added only 30 s prior to assay (11.6%) is markedly lower tan tat observed wen is coincubated wit te drug over te 60min period (76.1%). Tis suggests tat reduction of te redoxactive cysteine residues witin te disulfidebinding site of te enzyme is essential for inibition. In contrast to Triostam, te pentavalent analogue Pentostam is not inibitory, implying tat te antimony must be in te trivalent oxidation state to cause inibition. However, inibition by Triostam, potassium antimony tartrate or antimony tricloride (76.1 %, 84.6% and 83.7 % inibition, respectively) differs from stibocaptate (no inibition) despite all containing antimony in te trivalent state. Tis may be due to te stabilities of te respective complexes; Triostam and potassium antimony tartrate are complexes of SbIII wit organic acids (gluconate and tartrate, respectively) wile stibocaptate is te S,Sdiester of two atoms of SbIII wit tree molecules of 2,3captosuccinate exasodium salt. Complexes of eavy metals and ditiols are known to ave ig stability constants [25], wic would account for te relative ineffectiveness of stibocaptate. Tese results suggest tat te concentration of uncomplexed SbIII may be te significant factor in te extent of enzyme inibition. Bot L. donovani trypanotione reductase and uman erytrocyte glutatione reductase are inibited by Triostam in a timedependent manner (Fig. 2, A and B respectively). In bot cases, inibition occurs in two stages, te first being dependent upon inibitor concentration, te rates of wic are first order and pass troug 100% activity on extrapolation to zero time, wile te second stage is apparently independent of inibitor, tese rates being of te same magnitude as tose observed for inibition by alone. Tese data would be compatible wit a slow equilibrium formation between active enzyme/free inibitor and an inactive enzymeibitor complex. Te most apparent difference between te two enzymes is tat, at any concentration of inibitor, te degree of inibition at equilibrium is iger for trvvanotione reductase tan glutatione reductase. 111 C,. I vl x 4 aj z b I 1/ [Triostam] pm,, [Triostam] (pm) Fig. 2. Inibition of trypanotione reductase and glutatione reductase by Triostam. (A) Trypanotione reductase (7.2 nm), was incubated at 25 C wit 150 pm for 10 rnin followed by te addition of Triostam at time = 0 at a concentration of (0) none, (0) 5 pm, (V) 10 pm, (V) 20 pm, (0) 50 pm, (B) 100 pm. Aliquots were removed at intervals and assayed as described in Materials and Metods. Residual activity is expressed as a percentage of te activity at zero time. (B) Glutatione reductase (3.6 nm) was incubated as above and assayed using glutatione disulfide as substrate. (C) Net rate of inibition of trypanotione reductase (0) and glutatione reductase (0) as a function of Triostam concentration. Te inset sows a doublereciarocal renlot.

6 Cunningam and Fairlamb (Eur. J. Biocern. 230) bp v z 3 d 10 ' 1 Fig. 3. Protection of trypanotione reductase from inibition by Triostam wit MelT. Enzyme, initial concentration 300 nm, was incubated at 25 "C for 10 min wit 150 pm followed at zero time by te addition of: (0) buffer, (0) 18 pm MelT, (0) 50 pm Triostam, (7j 18 pm MelT + 50 pm Triostam. Aliquots were removed at intervals and residual activity determined by 50fold dilution into assay buffer containing (150 pmj and trypanotione disulfide (50 pm). After correction for inactivation due to alone, a plot of te net observed rate of inactivation (kobs) against inibitor concentration (Fig. 2C), sows a linear relationsip over te range of Triostam examined. Linear regression on tese data, wic, witin experimental limits, pass troug te origin, suggests tat tere is no appreciable formation of a noncovalent Micaelistype enzymelinibitor complex prior to te addition of substrate [33, 341. Te kos values at any cosen inibitor concentration are not markedly different between trypanotione reductase and glutatione reductase. A doublereciprocal plot of llnet k,,, against l/[triostam] (Fig. 2C, inset) yields a straigt line wic witin experimental limits passes troug zero indicating tat inibitor constants (K,) can not be obtained. Te fact tat reduction of te enzyme wit is required for inibition suggests tat protein sulfydryl groups, probably te redoxactive cysteine residues, are te target for inibition by Triostam. We confirmed tis by examining te ability of te competitive inibitor MelT to protect trypanotione reductase from inibition by Triostam (Fig. 3). In tis experiment, a ig concentration of enzyme was employed to permit a 50fold dilution 30 s prior to addition of substrate in order to dissociate te majority of te trypanotione reductasemelt complex. An initial MelT concentration of 18 pm was used, equal to twice te K, of 9 pm determined for te leismania enzyme (data not sown), suc tat, following dilution, inibition due to MelT would be quantitatively insignificant (1.5 %). As MelT is a simple competitive inibitor of te disulfidebinding site, coincubation wit Triostam sould result in lower inibition tan tat obtained wit Triostam alone, if te target of te antimonial is witin te disulfide binding site. Clearly MelT does protect trypanotione reductase from inibition, confirming te site of Triostam action. A ratio of te net observed rates of inibition due to Triostam in te presence or absence of MelT, klbs and kubs respectively, can be derived as k:bslko5 = ll(1 + ilk,) were i and K, represent MelT concentration and its inibitor constant, respectively. From Fig. 3, we obtain values for klbq and k,, of 3.25X103 si and 8.84X10' s', respectively, from wic a K, of 10.5 pm can be calculated, in good agreement wit our previously determined value of 9 pm. B R v 5 3 m 3 9 s Ql ffi I Fig. 4. Reversal of Triostam inibition of trypanotione reductase. (A) Reversal by dilution. Enzyme, final concentration 7.5 nm, was incubated at 25 C wit 150 pm for 10 min followed at time zero by te addition of: (0) buffer, (0) 5 pm Triostam, (7) 20 pm Triostam followed by (V) a 10fold dilution at time = 20 min. (B) Reversal by 2,3captopropanol. Enzyme, final concentration 5.9 nm, was incubated at 25 C wit 150 pm for 10 min followed at time zero by te addition of: (0) buffer, (V) 50 pm Triostam followed at time = 30 min by 100 pm 2,3captopropanol (7). Aliquots were removed at intervals and assayed as described in Materials and Metods. Activity is expressed as a percentage of te activity at time zero. Inibition of L. donovani trypanotione reductase by Triostam is readily reversible by dilution of te inibitor in a timedependent manner wic mirrors te initial inibition pase (Fig. 4A). Inibition by 50 pm Triostam was allowed to proceed for 20 min after wic te remaining sample was diluted 10 fold (final [Triostam] = 5 pm). Te initial kobs of reactivation, 2.7X103 s', is approximately 30% of te initial kobr of inibition, 8.7X103 SKI. However, complete recovery of activity to tat observed in te control treated wit 5 pm Triostam was not acieved. Addition of a twofold excess of 2,3captopropano1 (100 pm) to enzyme inibited by Triostam also results in rapid reactivation, again resembling te initial inibition, presumably as a consequence of sequestration of te antimony by te ditiol (Fig. 4B). Greater reactivation is observed in te latter case, activity returning to tat of te control value after 30 min. Te initial kos of reactivation is faster tan tat observed for dilution, being approximately 60% of tat of te inibition kob>. A twofold excess of te ditiols ditiotreitol and 2,3captosuccinate ave a similar effect to 2,3captopropanol, wilst neiter diydrotrypanotione, glutatione nor cyste

7 466 Cunningam and Fairlamb (Eur. J. Biocem. 230) 10 0 LO Fig. 5. Te effect of diydrotrypanotione on 'lkiostam inibition of trypanotione reductase. Trypanotione reductase (6.1 nm) was incubated at 25 C wit 150 m4 for 10 min followed by te addition of: (0) buffer, (0) 20 pm diydrotrypanotione, (0) 10 pm Triostam, ). ( 20 pm diydrotrypanotione followed immediately by 10 pm Triostam. Aliquots were removed at intervals and assayed as described in Materials and Metods. Residual activity is expressed as a percentage of te respective control activities at zero time. For clarity, te line for (U) diydrotrypanotione plus Triostam as been omitted. ine result in enzyme reactivation (data not sown). Tis may reflect iger stability constants of antimonials wit vicinal ditiols relative to eiter monotiols or to distal ditiols suc as diydrotrypanotione, as as been sown wit trivalent arsenicals Notably, a twofold excess of diydrotrypanotione added eiter prior to Triostam (Fig. 5) or after extensive inibition (as in Fig. 4B) neiter protects nor reverses enzyme inibition. Tis contrasts wit our previous work wic demonstrated tat diydrotrypanotione is capable of eiter protecting or reversing inibition of trypanotione reductase by te trivalent arsenical melarsen oxide by sequestering te arsenical as a stable covalent adduct, MelT [2S]. Furtermore, using HPLC analysis conditions were MelT formation is readily demonstrable (evidenced by a sift in retention time of te diydrotrypanotione moiety, Fig. 6, cromatograms b and c), neiter Triostam nor antimony tricloride appear to cause any suc canges (Fig. 6, cromatograms e and f, respectively). Te cromatograms sown in Fig. 6 (cf), represent incubation of 100 FM diydrotrypanotione wit 500 pm of te relevant antimonial or arsenical. Similar experiments using 50 pm antimonial or arsenical yielded identical results wit te antimonials, wilst wit melarsen oxide, equimolar amounts of diydrotrypanotione and MelT were observed. In contrast, te apparent adduct observed wit sodium arsenite, labelled X (Fig. 6, cromatogram d), could no longer be detected, suggesting tat te melaminopenyl moiety of melarsen oxide is important for stability of te adduct. Te above data, togeter wit our previous work wic suggests tat trivalent arsenicals interact covalently wit te redoxactive cysteine residues, prompted us to investigate te effect of Triostam on te visible absorption spectra of L. donovani trypanotione reductase (Fig. 1). As stated above, te enzyme exibits oxidized and reduced spectra caracteristic of a disulfidecontaining flavoprotein (trace A and B, respectively). Altoug te spectrum of te reduced enzyme is itself stable over time, subsequent addition of SO pm Triostam causes considerable timedependent canges in te absorption spectrum. Te absorbance at bot 463 nm and 530 nm decreases wit time (traces CF), until a stable spectrum is obtained after some 10 min, again suggestive of an equilibrium process. Fur 1 E I I x Fie.6. HPLC analvsis of te effects of antimonials and arsenicals ondiydrotrypanotione. Trypanotione disulfide (final concentration 100 pm) was incubated wit te following: trace a, no addition; trace b, 150 pm plus trypanotione reductase (0.3 U/rnl), te decrease in absorbance at 340 nm was followed until completion ten, to aliquots of tis sample, te following were added to 500 pm: trace c, rnelarsen oxide; trace d, sodium arsenite; trace e, Triostam; trace f, antimony tricloride. Equimolar amounts of trypanotione were analysed (2 nmol). ter addition of Triostam, to a final concentration of 100 pm after 40 min, again results in timedependent canges in te absorption spectrum until a new apparent equilibrium is reaced after approximately a furter 10 min (trace G). Interestingly, upon reacing tis apparent equilibrium, te spectrum exibits a broad longwavelengt absorbance band centred on approximately 650 nm extending to 800 nm. A similar band as been observed for glutatione reductase wen te intercange tiol is alkylated wit iodoacetamide and a ig concentration of H,O, added [35]. Te loss of te absorbance at 530 nm, caracteristic of a cargetransfer complex between te FAD and te proximal activesite cysteine, implies tat tis residue is interacting wit te antimonial. Previous work on bot C. fasciculata and T congolense trypanotione reductases as sown tat addition of iodoacetamide results in essentially complete preferential alkylation of te intercange tiol [22, 231. Tese points suggest tat te antimonial covalently interacts wit bot activesite cysteine residues to form a ditiostibane adduct. Te observed spectral canges are also consistent wit te direct reduction of FAD to FADH, [23, 241. We were unable to detect any measurable increase in te rate of oxidation upon incubation of trypanotione reductase (70 nm) wit and Triostam (200 pm) under aerobic conditions (data not sown). Tus reoxidation of FADH, to FAD by molecular oxygen and subsequent redox cycling is not stimulated by Triostam. Moreover, unlike te C. fasciculata enzyme [24], oxidation of was not stimulated by melarsen oxide, despite te inibition kinetics being oterwise similar (data not sown). Tis suggests tat binding of tese inibitors as different effects on te redox environments of te FAD groups in te respective enzymes. DISCUSSION Te pysical and kinetic properties of L. donovani trypanotione reductase are similar to tose of previously caracterised trypanotione reductases, in particular te ig specificity for trypanotione disulfide as substrate compared to glutatione di

8 Cunningam and Fairlamb (Eul: J. Biocem. 230) 467 sulfide (see review [12]). Te enzyme exibits te visible absorption spectra caracteristic of flavoprotein disulfide oxidoreductases, a family wic includes mercuric reductase and lipoamide deydrogenase in addition to glutatione reductase [36]. Te catalytic efficiency, k,,jk,,, of 5.OX1O6 M' s', is very similar to tose of te oter patogenic parasites T. cruzi [21] and 7: congolense [22], all of wic are 23fold lower tan tat observed for te insect parasite C. fasciculata enzyme [23]. In 1943, Goodwin and Page [9] proposed tat reduction of pentavalent antimonials to te trivalent form by te ost may be essential for teir antileismania1 activity. Tis is consistent wit te observations tat L. donovani, L. tropica and L. mexicana amazonensis promastigotes are all sensitive to trivalent sodium stibogluconate (Triostam) but insensitive to te pentavalent form (Pentostam), wilst L. donovani and L. tropica amastigotes witin macropages are sensitive to Pentostam in vitro [8, 371. Trivalent antimonials inibit bot trypanotione reductase and uman erytrocyte glutatione reductase in vitro, wilst bot enzymes are insensitive to te pentavalent form. Te mecanism appears similar in many respects to tat found for trivalent arsenicals [24]. Inibition occurs witin te disulfidebinding site, wit prior reduction of te redoxactive disulfide bridge by being an essential prerequisite. Spectral evidence suggests tat te cargetransfer tiol is involved, despite te fact tat tis cysteine is te least accessible of te two, bot structurally [13] and cemically [23]. In turn, tis suggests tat te intercange tiol is likely to react wit te antimonial first. Tus, we propose tat interaction of te free antimonial wit te intercange tiol generates a transient monotiostibane adduct. Tis is followed by a rapid, essentially instantaneous, intramolecular rearrangement, binding te cargetransfer tiol to form a stable ditiostibane complex encompassing bot redoxactive cysteine residues. In tis mecanism, te ratelimiting step is te initial formation of te monotiostibane complex. Tis contrasts wit te organic arsenicals, were formation of te monotioarsane adduct is essentially instantaneous and subsequent rearrangement to form te ditioarsane complex appears ratelimiting [24]. Furtermore, te 13fold faster rate of inibition (k,,,,,) of trypanotione reductase over glutatione reductase by trivalent arsenic substituted wit a bulky melaminopenyl group (melarsen oxide) could be attributed to te relatively narrower confines of te disulfidebinding site in te mammalian enzyme [13, 171. In contrast, te rates of inibition (kobs) of trypanotione reductase and glutatione reductase are not markedly different for any given concentration of Triostam. Tis is consistent wit tere being little or no steric indrance in te rearrangement of te less bulky monotiostibane complex in te formation of te ditiostibane complex. Te involvement of oter activesite residues, suc as His461, in te formation and/or stabilisation of te enzyme/antimonial complex cannot be excluded at present. Xray crystallograpy of T. cruzi trypanotione reductase (inibited in a similar manner to te L. donovani enzyme) exposed to Triostam may sed furter ligt on tis question. Trivalent arsenicals form stable complexes wit diydrotrypanotione, bot in vitro and witin intact cells [25]. Under identical conditions were tese complexes are readily observed, we ave been unable to demonstrate any equivalent antimoniay diydrotrypanotione interactions. 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