FEBS Letters 584 (2010) 3376–3380

journal homepage: www.FEBSLetters .org

Kinetic and thermodynamic properties of two barley thioredoxin h isozymes,HvTrxh1 and HvTrxh2

Kenji Maeda a,1, Per Hägglund a, Olof Björnberg a, Jakob R. Winther b, Birte Svensson a,*

a Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmarkb Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark

a r t i c l e i n f o

Article history:Received 30 April 2010Revised 10 June 2010Accepted 15 June 2010Available online 23 June 2010

Edited by Peter Brzezinski

Keywords:ThioredoxinDithiol/disulfide exchangeTryptophan fluorescenceRedox potentialThiol pKa

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.06.028

Abbreviations: E�0 , standard redox potential; EcTrxbarley thioredoxin h isozyme 1; HvTrxh2, barley thiodoacetamide; NTR, NADPH-dependent thioredoxinboxyethyl)phosphine; Trx, thioredoxin

* Corresponding author. Fax: +45 4588 6307.E-mail address: bis@bio.dtu.dk (B. Svensson).

1 Present address: EMBL Heidelberg, MeyerhofsGermany.

a b s t r a c t

Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show dis-tinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measure-ment of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibitsredox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamicproperties were readily determined. The reaction constants were �60% higher for HvTrxh1 thanHvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the activesite Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacet-amide titration, but showed �70% higher reactivity in HvTrxh1, suggesting significant functionaldifference between the isozymes.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Thioredoxin (Trx) is a ubiquitous protein disulfide reductase of�12 kDa [1] containing the so-called Trx-fold with a commonbababba topology [2]. The Trx superfamily includes proteins withdiverse functions and redox potentials ranging from Trx and glut-aredoxin acting as reductants in the cytosol, to the eukaryoticendoplasmatic protein disulfide isomerase and prokaryotic peri-plasmic DsbA both involved in disulfide bond formation [3].

The cysteines in the active-site motif, Trp-CysN-Gly-Pro-CysC,form an intramolecular disulfide bond in oxidized Trx. Trx receivesreducing equivalents from NADPH-dependent Trx reductase (NTR)or, in plant chloroplasts, from ferredoxin-dependent Trx reductase[4]. The CysN thiol acts as a nucleophile and attacks target disulfidebonds to form a transient mixed disulfide; the buried CysC thiolcompletes the reaction by attacking CysN with formation of oxi-dized Trx and reduced target disulfide. The CysN pKa among Trx

chemical Societies. Published by E

, E. coli thioredoxin; HvTrxh1,ioredoxin h isozyme 2; IAM,reductase; TCEP, Tris(2-car-

traße 1, 69117 Heidelberg,

superfamily members ranges from >6.3 for Trx [5–8] to <4 forGrx [9] and DsbA [10].

Higher plants possess numerous Trx isozymes which aregrouped based on sequence similarity [11]. Trx h constitutes thelargest group of isozymes suggested to have distinct biologicalroles [12]. Trx h is proposed to be important for seed germinationby solubilising storage proteins and inactivating inhibitors of pro-teases and carbohydrate-degrading enzymes through disulfidereduction [13]. Barley seeds contain at least two Trx h and twoNTR isozymes (barley thioredoxin h isozyme 1 (HvTrxh1), barleythioredoxin h isozyme 2 (HvTrxh2), HvNTR1 and HvNTR2) withdistinct spatiotemporal distribution patterns representing one ofthe best characterized Trx systems from higher plants [14,15].Here, redox properties of the two barley Trx h isozymes are char-acterized and compared in details involving a novel approachbased on reaction with Escherichia coli thioredoxin (EcTrx).

2. Materials and methods

2.1. Protein production

Untagged and his-tagged HvTrxh1 and HvTrxh2 were producedas described [14,16]. The tag did not affect activity in a Trx reduc-tase assay (data not shown). EcTrx was from Promega (Madison,WI, USA). Protein concentrations were determined by amino acidanalysis.

lsevier B.V. All rights reserved.

Fig. 1. Reduction of bovine insulin by the barley Trx system. Rates of NADPHoxidation with 0.1 lM HvTrxh1 (circles) and HvTrxh2 (squares) in the presence of0.2 lM thioredoxin reductase are shown and fitted to the Michaelis–Mentenequation.

K. Maeda et al. / FEBS Letters 584 (2010) 3376–3380 3377

2.2. Insulin reduction assay

The reaction mixtures contained his-tagged HvTrxh1 orHvTrxh2 (0.1 lM) in 0.1 M potassium phosphate pH 7.0, 2 mMEDTA, 0.1 mg/ml BSA, 0.16 mM NADPH and insulin in a range ofconcentrations up to 260 lM. The reactions were initiated by addi-tion of HvNTR2 to 0.2 lM, and the oxidation of NADPH was mon-itored spectrophotometrically at 340 nm for 4 min. A backgroundDA340 of 0.0014 min�1 obtained in the absence of insulin was sub-tracted from the data which were fitted to the Michaelis–Mentenequation using Kaleidagraph (Synergy Software, Reading, PA, USA).

2.3. Fluorescence emission spectra of Trxs

The assay buffer (100 mM Hepes pH 7.0, 1 mM EDTA) was de-gassed, thoroughly purged with argon, and throughout the exper-iment exposure of Trxs to oxygen was avoided. Fluorescencespectra of 12 lM untagged Trx in the assay buffer (pre-incubatedfor at least 1 h in 100 lM Tris(2-carboxyethyl)phosphine (TCEP)for reduced Trx) were acquired using a Perkin–Elmer Lumines-cence Spectrometer LS55 with a thermostated single-cell. The exci-tation wavelength and emission wavelengths were 280 nm (5 nmslit width) and 300–400 nm (8 nm slit width), respectively.

2.4. Fluorescence Trx assay

Reduced Trx was obtained by incubating 60 lM Trx with1.0 mM TCEP in 100 lL for at least 1 h at RT. TCEP was removedusing a NAP-5 desalting column (GE Healthcare). When Trx wasomitted no significant fluorescence increase was observed for themixture of the desalted solution and the oxidized EcTrx (data notshown), confirming complete removal of TCEP. The temperatureof the Trx solutions and the cuvette was adjusted to 25 �C priorto assays. Assays at 25 �C were initiated by mixing reduced andoxidized Trxs to a final concentration of 6.0 lM each in a volumeof 150 ll. The initial value of fluorescence intensity was recorded1 min after mixing, with excitation and emission wavelengths of280 and 380 nm (8 nm slit width), respectively, and the fluores-cence intensity was followed with 2–5 min intervals.

2.5. Alkylation kinetics

His-tagged Trxs (12 lM) were pre-incubated with 0.5 mM TCEPin 5 mM HEPES pH 7.0, 50 mM NaCl at RT for at least 1 h. Alkyl-ation of 4.0 lM reduced Trx was performed in duplicates at RTwith 20 lM iodoacetamide (IAM) in 1.0 mM EDTA, 200 mM NaCland 30 mM MES (pH 5.5–6.6), HEPES (pH 7.0–7.8), and Tris–HCl(pH 8.0–8.6). The reactions were quenched at appropriate timepoints by adding 40% acetic acid (50 ll) to the assay mixture(150 ll). Unmodified and carbamidomethylated Trxs were sepa-rated and quantified using a C18 RP-HPLC column on an ICS-3000 Ion Chromatography System (Dionex, Sunnyvale, CA, USA)at 30 �C. The column was pre-equilibrated with solvent A (0.1% tri-fluoroacetic acid) and proteins were eluted by a linear 37–54% gra-dient with solvent B (0.1% trifluoroacetic acid, 90% acetonitrile) for25 min at 1.0 ml min�1. Using CurveExpert v. 1.3 (D.G. Hyams, Hix-son, TN, USA) the second order reaction constants, k, were obtainedby fitting to Eq. (1):

kt ¼ 1½Trx�0 � ½IAM�0

ln½Trx�½IAM�0½IAM�½Trx�0

ð1Þ

k values were fitted to the Henderson–Hasselbalch equation (Eq.(2)):

k ¼ kmax

1þ 10ðpKa�pHÞ ð2Þ

3. Results

3.1. Enzymatic properties

We previously reported that HvTrxh1 and HvTrxh2 with 51%sequence identity are reduced by HvNTR2 with similar kineticparameters � Km of 1.12 ± 0.04 and 1.29 ± 0.25 lM and kcat of3.26 ± 0.09 and 2.98 ± 0.16 s�1 for HvTrxh1 and HvTrxh2, respec-tively [15]. Here, reduction of bovine insulin by HvTrxh1 andHvTrxh2 was assayed in a coupled reaction with HvNTR2 as elec-tron donor. Measurement of NADPH oxidation rates (340 nm)yielded limiting rates of 0.0395 ± 0.00079 and 0.0334 ± 0.001D340/min, corresponding to turnovers of 1.1 and 0.90 disulfidesper second for HvTrxh1 and HvTrxh2, respectively (Fig. 1). Appar-ent Km values of 26.1 ± 1.8 and 37.8 ± 4.8 lM obtained for HvTrxh1and HvTrxh2, respectively by fitting to the Michaelis–Mentenequation were comparable to the Km of 11 lM found for EcTrx[17]. HvTrxh1 thus showed significantly higher catalytic efficiencythan HvTrxh2 with kcat/Km being 0.41�105 and 0.24�105 s�1 M�1,respectively. This difference was not rate-limited by the reductase(data not shown) and can be compared to the kcat/Km ratio of1 � 105 M�1 s�1reported for EcTrx with insulin as substrate [17].

3.2. Oxidoreduction kinetics and thermodynamics

To elucidate the oxidoreduction kinetics and thermodynamicsof HvTrxh1 and HvTrxh2, EcTrx was employed both as electron do-nor and acceptor in the electron transfer reactions:

EcTrxred þHvTrxh1ox ðor HvTrxh2oxÞ$ EcTrxox þHvTrxh1red ðor HvTrxh2redÞ

The reactions can be continuously monitored as the fluores-cence intensity of EcTrx is redox-sensitive owing to the non-conserved tryptophan at position �4 from CysN [18]. Indeed, com-plete disulfide reduction by TCEP increased the fluorescence inten-sity >fivefold for EcTrx (Fig. 2A), but had only minor effect onHvTrxh1 and HvTrxh2 which only are 22% and 23% sequence iden-tical to EcTrx, respectively, and lack this tryptophan (Fig. 3). Whenoxidized EcTrx (6.0 lM) and equimolar amounts of the reducedHvTrxh1 or HvTrxh2 were mixed, the fluorescence intensity in-creased due to EcTrx reduction and reached steady state after �5and �10 h, respectively (Fig. 2B). The increases in fluorescence

Fig. 2. Fluorometric measurement of redox reactions between Trxs. (A) Fluorescence emission spectra for HvTrxh1, HvTrxh2 and EcTrx in the oxidized (dashed line) andreduced (solid line) state. (B) Time-course of emission intensity for mixtures containing oxidized EcTrx and reduced HvTrxh1 or HvTrxh2. (C) Initial changes in thefluorescence intensity for mixtures containing oxidized EcTrx and reduced HvTrxh1 (filled diamonds), oxidized EcTrx and reduced HvTrxh2 (filled triangles), reduced EcTrxand oxidized HvTrxh1 (open diamonds), reduced EcTrx and oxidized HvTrxh2 (open triangles). (D) Time-course for the molar concentrations of the individual reactantscalculated and shown for the same reactions as in (C).

Fig. 3. Sequence alignment of EcTrx, HvTrxh1 and HvTrxh2. The non-conserved tryptophan residue providing redox-sensitive fluorescence properties of EcTrx and the twoconserved catalytic cysteine residues are highlighted.

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intensities corresponded to reduction of 2.4 and 2.8 lM EcTrx byequimolar amounts of HvTrxh1 and HvTrxh2, respectively. Thestandard redox potentials (E�0) for HvTrxh1 and HvTrxh2 were cal-culated to –260 and –263 mV, respectively, using the obtainedequilibrium concentrations of each Trx species, the reported E�0

of –270 mV for EcTrx [19] and the Nernst equation (Eq. (3));

E�0ðHvTrxh1=2Þ ¼ E�0ðEcTrxÞ � RTnF� ln K ð3Þ

in which K is the equilibrium constant.The initial reaction rates were determined for equimolar mix-

tures of oxidized EcTrx with reduced HvTrxh1 or HvTrxh2 andfor reduced EcTrx with oxidized HvTrxh1 or HvTrxh2 (Fig. 2C).Data were acquired only until up to �10% reactants were con-sumed to minimize influence from the reverse reactions. Basedon the changes in fluorescence intensities, the molar concentra-tions for the remaining reactants were calculated at each timepoint (Fig. 2D). The second order reaction constants for the oxida-tion (k2) and reduction (k1) of HvTrxh1 were 13 and 15 M�1s�1,respectively, while both values for HvTrxh2 were 8.2 M�1s�1, thusshowing a significant kinetic difference between the two isozymes.

Moreover, the kinetic data enabled calculation of E�0 (K = k1/k2)without exposing Trxs for prolonged incubation in contrast tothe above described equilibrium-based approach. This approachassigned very similar E�0 values of –268 and –270 mV for HvTrxh1and HvTrxh2, respectively, also comparable to the values obtainedby the equilibrium-based approach. The standard deviation of trip-licate kinetic-based E�0 determination was 1 mV for both Trxs.

3.3. pH-titration of the CysN thiol

To understand the background for the observed kinetic varia-tions between HvTrxh1 and HvTrxh2, alkylation rates were deter-mined for CysN thiol of reduced HvTrxh1 and HvTrxh2 at pH 5.5–8.6. Trxs were treated with IAM for various time lengths, and theCysN-carbamidomethylated and unmodified Trxs were separatedby HPLC [20]. Trxs appeared in a single peak when IAM-treatedat pH 5.5, while a second peak eluted at a shorter retention timewhen pH was increased (Fig. 4A). The carbamidomethylated andunmodified Trx fractions were quantified, and second order reac-tion constants obtained for each pH value (Fig. 4B). The reactionconstants were �70% higher for HvTrxh1 than HvTrxh2 in the en-

Fig. 4. pH-titration of CysN in HvTrxh1 and HvTrxh2. (A) Chromatographic separation of carbamidomethylated and unmodified HvTrxh1 and HvTrxh2 after treatment of4.0 lM Trx with 20 lM IAM at various pH values for 40 s. (B) Fitting of the determined carbamidomethylated Trx concentrations at different time points to an equation forsecond order reaction of HvTrxh1 at pH 7.8 (filled square) and pH 7.0 (filled triangle), and HvTrxh2 at pH 7.8 (open square) and pH 7.0 (open triangle). (C) Obtained secondorder reaction constants at various pH values for HvTrxh1 (filled diamonds) and HvTrxh2 (open diamonds) fitted to Henderson–Hasselbalch equation.

K. Maeda et al. / FEBS Letters 584 (2010) 3376–3380 3379

tire pH range and showed a single transition for both Trxs (Fig. 4C).Fitting the data to the Henderson–Hasselbalch equation gave thesame apparent pKa of 7.6 for CysN in both isozymes (Fig. 4B).

4. Discussion

To compare the oxidoreduction properties of HvTrxh1 andHvTrxh2, we developed a novel approach based on redox reactionsbetween these proteins and EcTrx. The present kinetics-based ap-proach for E�0 assignments has the advantage relative to equilib-rium-based approaches [21] in that it circumvents prolongedincubation time that may cause precipitation or oxidation of pro-teins. Furthermore, a very low standard deviation of E�0 measure-ments was achieved compared to a more traditional methodusing DTT as a reference [22]. This strategy can be valuable forother comparative studies of redox-active proteins, e.g. in muta-tional analyses as thermodynamic and kinetic consequences ofthe mutation can be analyzed in a single experiment. The redox-dependence of EcTrx tryptophan fluorescence is transferable toother Trx superfamily members by introducing a tryptophan resi-due at the active-site by site-directed mutagenesis as previouslydemonstrated for E. coli DsbD [23], providing a possibility to applythe novel strategy for proteins with diverse thermodynamic oxido-reduction properties.

The present study has shown that HvTrxh1 and HvTrxh2 havehighly similar E�0, HvTrxh2 moreover having a value identical tothat of the reference protein EcTrx (�270 mV). The HvTrxh1 andHvTrxh2 similarities extend to the finding of identical apparentpKa 7.6 for CysN by IAM alkylation. From investigations involvingboth NMR and observation of the thiolate absorbance at 240 nm,there is a general consensus of a single pKa around 7.0 for EcTrx[8]. However, IAM alkylation of EcTrx showed two transitions withapparent pKa of 6.7 and 9 [5]. These two transitions in EcTrx wereconfirmed by iodoacetate alkylation and even a lower pKa of ca 3was observed [20]. The presence of multiple transitions beyondthe pH range analyzed here (5.5–8.6) cannot be excluded, althoughthe pH-titration curves for alkylation of HvTrxh1 and HvTrxh2with a single transition at pKa 7.6 is indeed different from that ofEcTrx.

Even though similar values for E�0 and CysN pKa were obtained,HvTrxh1 showed increased reactivity in terms of both IAM alkyl-ation and kinetics of EcTrx oxidoreduction. These findings suggestvariation in the local environment surrounding the CysN thiol be-

tween the barley Trx h isozymes, in spite of high conservation insequence and structure around the active-site residues [24].

Acknowledgements

Mette Therese Christensen and Karina Rasmussen are acknowl-edged for technical assistance. Anne Blicher is thanked for per-forming amino acid analysis. The Danish Technical ResearchCouncil (STVF, Grant No 26–03–0194) and the Carlsberg Founda-tion are acknowledged for financial support. Dr. Christine Finnieis thanked for careful reading of the manuscript. K.M. received aPh.D. scholarship from the Technical University of Denmark.

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