Biochimica et Biophysica Acta 1794 (2009) 367–373

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Kinetic analysis of salting activation of a subtilisin-like halophilic protease

Débora N. Okamoto a, Marcia Y. Kondo a, Jorge A.N. Santos a, Sawa Nakajima b, Kazumi Hiraga b, Kohei Oda b,Maria A. Juliano a, Luiz Juliano a, Iuri E. Gouvea a,⁎a Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo. Rua Três de Maio 100, 04044-20 São Paulo, Brazilb Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan

⁎ Corresponding author. Departamento de Biofísica,UNIFESP, Rua Três de Maio, 100 São Paulo – 04044-020,

E-mail addresses: gouvea@biofis.epm.br, iurig@yaho

1570-9639/$ – see front matter © 2008 Published by Edoi:10.1016/j.bbapap.2008.10.017

a b s t r a c t

a r t i c l e i n f o

Article history:

The secreted extracellular Received 23 September 2008Received in revised form 13 October 2008Accepted 23 October 2008Available online 12 November 2008

Keywords:PeptidaseSerine proteaseProton inventory

subtilase SR5-3 from Halobacillus sp. bacterium, isolated from the high-saltenvironment of Thai fish sauce, was utilized as a model halophilic serine protease. The dependence of saltactivation on the size and structure of substrates was evaluated assaying the enzyme with Suc-AAPF-MCAand with the Fluorescence Resonance Energy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp. Solvent isotopeeffects (SIE) and the thermodynamic parameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease in the presence of salts were also performed. All the obtained resultssupport the notion that the salting out effect is responsible for the halophilic character of SR5-3, and themagnitude of its hydrolytic activity is mainly derived from the improvement of catalytic and/or interactionsteps depending on the nature and size of the substrates, principally if they occupy the substrate primesubsites.

© 2008 Published by Elsevier B.V.

1. Introduction

Halophilic (from Greek hals= salt, phil=affinity) enzymes areobtained from microorganisms isolated from environments with highsalt concentration (minimumvalue defined as 2.5MNaCl for optimumgrowth) [1–2]. Such microorganisms are divided into three groups:aerobic halophilic Archaea, anaerobic halophilic methanogenicArchaea, and halophilic bacteria [1]. The best characterized halophilicenzymes are those from the first group that balances the extremeosmotic pressure of hypersaline environments by intracellularaccumulation of inorganic salts, while the halophilic bacteriaaccumulatemainly neutral organic solutes [3]. The halophilic enzymesperform identical functions as their non-halophilic counterparts butrequire salt, usually NaCl or KCl in the 1–4 M range, to keep theiractivity, solubility and stability [2–4]. The electrostatic repulsioncaused by an abundance of negative charges on the surface ofhalophilic proteins can be neutralized by the salt counter ionsresulting in a more stable folded structure [5–6]. As a consequence,the halophilic enzymes can assume a balance between flexibility toenable catalytic property, and rigidity to avoid denaturation by saltexcess [4].

The stabilization and activation of proteases by kosmotropic saltshave also been described in several non-halophilic organisms thatrange from viral [7–10] to human proteases [11–12]. The kosmotropic

Escola Paulista de Medicina –

Brazil. Fax: +55 11 5575 9617.o.com (I.E. Gouvea).

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salts act on proteins by the canonical salting out mechanism, resultingin enzyme activation since in this process salt acts by not coming intocontact with the enzyme, but being excluded from the protein [11–13].In the halophilic enzymes it is proposed that hydrated salt ions are incontact on their surface at higher local concentrations than in thesurrounding solution by specific interactionswith the surface carboxylgroups, and the water molecules bind to halophilic proteins throughhydrated associated salt [4]. The molecular mechanisms and thekinetics of hydrolytic activities of proteases activated by salt andderived from halophilic microorganisms are not well defined. Thephysical chemistry driving adaptation of proteases to extremeenvironments is a useful tool for the understanding of the mechan-isms involved in protein–solvent interactions. In addition, there are asubstantial number of identified halophilic proteases [14–16] that areof interest for biotechnological and industrial applications [17].

In the present work we used, as a model of a halophilic serineprotease, the secreted extracellular subtilase SR5-3 from Halobacillus sp.bacterium. This halophilicmicroorganismwas isolated from the high-saltenvironment of prepared Thai fish sauce, which is a condiment preparedfrom fish in concentrated brine [18]. We present data for the hydrolyticactivity of subtilase SR5-3 on synthetic fluorogenic peptides in thepresence of different cations, and compare the effects of kosmotropic andneutral anions of theHofmeister series. The dependence of salt activationon the size and structureof the substrateswas also evaluated assaying theenzyme with Suc-AAPF-MCA and with the Fluorescence ResonanceEnergy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp where glutami-nyl-[N-(2,4-dinitrophenyl)-ethylenediamine] (Q-EDDnp) is the fluores-cence quencher and ortho-aminobenzoic acid (Abz) is the fluorescentgroup. The FRET peptide allowed exploring the role of prime sites (i.e.

368 D.N. Okamoto et al. / Biochimica et Biophysica Acta 1794 (2009) 367–373

carboxy-terminal to site of hydrolysis) of the substrates [19] on the saltactivation process. In addition, we did proton inventory, i.e., the kineticstudies of solvent isotope effects (SIE) in a mixture of H2O and D2O [20–22] in the presence of salts, and also determined the thermodynamicparameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease.

2. Material and methods

2.1. Materials

Anhydrous dimethyl sulfoxide (DMSO), heavy water with 99.9%deuterium content, and anhydrous methanol were purchased fromAldrich Chemical Co. All buffer salts were reagent-grade andpurchased from Aldrich, Fisher, or Sigma Chemical Co.

2.2. Peptides

Suc-AAPF-MCA (MCA, 7-amino-4-methylcoumarin) was a gener-ous gift from Dr. K. Takada, Peptide Institute Inc. (Osaka, Japan).The FRET peptides were synthesized by solid-phase synthesis andpurified as earlier described [23–24]. The molecular mass andpurity of synthesized peptides were confirmed by amino acidanalysis and by MALDI-TOF using a Microflex-LT mass spectrometer(Bruker — Daltonics, Billerica, MA, USA). Stock solutions of peptideswere prepared in DMSO, and their concentrations were measuredspectrophotometrically using a molar extinction coefficient of17.300 M−1 cm−1 at 365 nm.

2.3. Solutions

Tris–HCl buffer solutions for the proton inventory studies wereprepared gravimetrically by mixing appropriate quantities of Tris–HClbuffers made in H2O or D2O at pL 8.0. The pD of deuterium oxidesolution was obtained from pH meter readings according to therelationship pD=pH (meter reading)+0.4 [25]. The fraction ofdeuterium in each buffer was calculated accounting for density,mass of the enzyme stock solution, and presence of buffer salts.

2.4. Enzymes

Halophilic protease from Halobacillus sp. SR5-3 was obtained aspreviously described [18]. Briefly, the Halobacillus sp. SR5-3 wascultivated in modified JCM 377 medium and the culture broth wascentrifuged at 10,000 rpm for 30min. The secreted enzymewas purifiedfrom the soluble fraction by ammonium sulfate precipitation and byaffinity chromatography using a Bacitracin-Sepharose 4 Fast Flowcolumn. The eluted fractions were assayed for proteolytic activity asdescribed below, and the purity of enzyme analyzed by SDS-PAGE wassimilar to previously described preparations [18]. SR5-3 enzyme proteinconcentrationwas estimated using the Bradford method using albuminas standard. Subtilisin Carlsberg was purchased from Sigma-Aldrich.

2.5. Salt influence on SR5-3 activity and stability

The influence of NaCl, NaOAc (sodium acetate), Na2SO4, NaH2PO4,sodium citrate, KCl, CaCl2, MgCl2 and NH4Cl on the catalytic activity ofSR5-3 was investigated over concentration range of 0.5–5 M. Theassays were performed measuring the initial velocity of hydrolysis of20 μM Suc-AAPF-MCA as substrate, at 37 °C in 50 mM Tris–HCl with(V), or without (V0), salt to obtain the ratio of V/V0 (reflecting theactivation by salt). All assays were performed at pH 8.0. The effect ofNaCl on SR5-3 stability was analyzed by measuring enzyme activityremaining after incubation in 0, 1.5 and 3 M NaCl at 37 °C for 48 h,under the same conditions described above. Subtilisin Carlsberg wasassayed in 50 mM Tris–HCl pH 8.0 in the presence of 1 mM CaCl2.

2.6. Kinetic measurements

Hydrolysis of MCA peptides was assayed in a Hitachi F-2500spectrofluorimeter, at 37 °C. Fluorescence changes were monitoredcontinuously at λex=380 nm and λem=460 nm. The enzyme concentra-tionswere chosen such that less than 5% of the substratewas hydrolyzedover the course of the assay. The reaction rate was converted intomicromoles of substrate hydrolyzed per minute based on a calibrationcurve obtained fromthe complete hydrolysis of eachpeptide.WhenFRETpeptideswere used the assay conditionwasmodified toλex=320 nmandλem=420 nm. The inner-filter effect was corrected using an empiricalequation as previously described [26]; furthermore, the concentration ofDMSO was kept below 1% to avoid inhibiting the enzyme activity thatoccurred at solvent concentration higher than 5%. In addition, thesubstrates were completely soluble in all used buffer conditions till atleast 2-fold of the Km values for their kinetics of hydrolysis.

The kinetic parameters Km and kcat with respective standard errorswere fit to the Michaelis–Menten equation using GraFit® software(Erithacus Software, Horley, Surrey, U.K.). Data was collected intriplicate, and the error values were less than 5% for each of theobtained kinetic parameters.

2.7. Determination of the substrate cleavage sites

The scissile bond of hydrolyzed peptides was identified by isolationof the fragments using analytical HPLC followed by determination oftheir molecular mass by LC/MS using an LCMS-2010 equipped with anESI-probe (Shimadzu, Japan).

2.8. The pH and pD dependence of specificity constant

The pH dependence of the hydrolysis rate constant was measuredat 25 °C in a two-component buffer comprised of 50 mM Tris and50 mM glycine, adjusted to the required pH by addition of 1 M NaOHor 1MHCl. In each pH the ionic strengthwasmaintained at 0.1 Mwithsodium chloride. The same methodology was used to determine theeffect of 3 M NaCl and 1 M Na2SO4 on SR5-3 pH profile experiments.The reactions were performed under pseudo first-order conditions ofhydrolysis of the substrates. The data was fitted with the GraFit®

software to the appropriate equation.

2.9. Temperature dependence of the specificity constant

Temperature dependence of the specificity constant (kcat/Km) wasdetermined as earlier described [27,28]. The hydrolysis of Suc-AAPF -MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 were monitored underpseudo first-order conditions ([S]bbKm) and Michaelis–Mentenkinetics, respectively, in Tris–HCl pH 8.0 buffer, either in absence orpresence of 1 M sodium sulfate or 3 M NaCl. Temperature correctionswere applied to Tris buffers according to (ΔpKa/Δt=−0.027). Activa-tion parameters were calculated from the linear plot of ln[(kcat/Km)/T]versus 1/T (Eq. (1)).

ln kcat=Kmð Þ=T½ � = ln R=NAhð Þ +ΔST=R−ΔHT=RT ð1Þwhere R is the gas constant (8.314 J mol−1K−1), T is the absolutetemperature, NA is the Avogadro number, h is the Plank constant,enthalpy of activation ΔH⁎=−(slope)×8.314 J mol−1, the entropy ofactivation ΔS⁎=(intercept−23.76)×8.314 J mol−1K−1. The free energyof activation ΔG⁎, was calculated from Eq. (2).

ΔGT =ΔHT−TΔST ð2Þ

2.10. Proton inventory experiments

Experiments were performed at pH 8.0 and equivalent pL inisotopic buffers at eleven different values of n ranging from 0 to 0.99 n

Table 1Models used for fitting proton inventory data as described by Enyedy and Kovach [22]

Obtained data Equation

TS1 Vn=VH(1−n+nØ1)TS1, solv Vn=VH(1−n+nØ1)ØS

n

2TS1 Vn=VH(1−n+nØ1)2

2TS1, solv Vn=VH(1−n+nØ1)2ØSn

TS1, TS2 Vn=VH(1−n+nØ1)(1−n+nØ2)TS1, TS2, solv Vn=VH(1−n+nØ1)(1−n+nØ2)ØS

n

TS, RS Vn=VH(1−n+nØ1)/(1−n+nØR)

369D.N. Okamoto et al. / Biochimica et Biophysica Acta 1794 (2009) 367–373

either in pseudo-first-order conditions or in Michaelis–Mentenkinetics, and performed at least in triplicate. The proton inventory(PI) methodology was performed using the protocol developed byEnyedy and Kovach [22] based on the fitting of the experimental datawith simplified forms of the Gross–Butler Eq. (3) that relates thedependence of a particular rate parameter to the atom fraction ofdeuterium, n, in the solvent mixtures:

Vn = V0 ∏TS

i1−n + nO=T

i

� �=∏RS

j1−n + nO=R

j

� �ð3Þ

where Vn and V0 are velocities (or rate constant) in a binary solventand inwater, respectively, n=atom fraction of deuterium, RS=reactantstate, ØR=RS fractionation factor, and ØT=TS fractionation factor. Alldata reductions using the simplified forms of this equation forhydrolytic enzymes (Table 1) were performed using the GraFit 3.3software (Erithacus Software Ltd., Middlesex, UK). The fractionationfactors were calculated from the best statistical results, measured by

Fig.1. Effect of salts on SR5-3 hydrolytic activity and stability. (A) Effect of NaCl concentration.of 20 µM Suc-AAPF-MCA by SR5-3 protease (●) and subtilisin Carlsberg ( ) in the absencedetermined after enzyme incubation in the absence (○) and presence of 1.5 (●) and 3.0 M (□Hofmeister anions series. SR5-3 activity was assayed in the presence of Sodium citrate (●),activity was assayed in the presence of NaCl (△), KCl (○), NH4Cl (●), MgCl2 (▾) and CaCl2 (

the reduced x2 and based on the goodness of fit and on a consistentand reliable mechanism.

3. Results

3.1. Effects of salts on halophilic enzyme activity

The effect of NaCl on SR5-3 activity was determined by measuringthe initial velocity of hydrolysis of Suc-AAPF-MCA, and indicated a 20-fold activation in 3.5 M NaCl. In contrast, the activity of subtilisinCarlsberg was salt insensitive in the same assay conditions (Fig. 1A).Besides activation, NaCl also increased SR5-3 stability upon incubationat 37 °C over 48 h, as shown in Fig. 1B.

In order to explore in more detail the peptidase activationprocess of SR5-3, we analyzed the effects of Hofmeister seriesanions in concentrations up to 1.5 M, with Na+ as the commoncation (Fig. 1C). SO4

2− and citrate were the most effective activatorswith a remarkable increase of almost 100-fold, whereas HPO4

2− wasless effective. The enzyme was also activated by cations (with Cl− ascommon anion), with Na+ and K+ being the most effective, followedby NH4

+ and Mg2+. Curiously, Ca2+ presented biphasic behavior,activating SR5-3 up to 1 M and inhibiting its activity at higherconcentrations (Fig. 1D).

The effect of salts on the pH dependence of SR5-3 peptidase activitywas determined over the pH range of 6.3–10.5 (Fig. 2). Optimalhydrolysis occurred above pH 8.0 in all assayed conditions with asimple sigmoid pH-rate profile. The obtained pKe values were 6.8±0.3,7.2±0.2 and 6.9±0.2 for the profiles in absence of salt, in presence of3MNaCl (neutral salt), and1MNa2SO4 (kosmotropic salt), respectively.

Activation factors (V/V0) were determined bymeasuring the initial velocity of hydrolysisor presence of NaCl up to 3.5 M. (B) Effect of NaCl on stability. The SR5-3 activity was) NaCl at 37 °C. The assay conditions are described in Materials and methods (C) Effect ofNa2HPO4 (▾), Na2SO4 (□), Sodium Acetate (■) and NaCl (△) (D) Effect of cations. SR5-3□).

Fig. 2. pH-kcat/Km profile for the reaction of SR5-3 protease with the substrate Suc-AAPF-MCA in the absence of salt (○) and in the presence of 3 M NaCl ( ) or 1 M Na2SO4

(●). Relative activities were calculated as percentage of the highest kcat/Km valuesobtained in each assay condition. The hydrolysis conditions are described in Materialsand Methods.

Fig. 3. Proton inventory of the specificity constant k (kcat/Km) for the hydrolysis of (A)Suc-AAPF-MCA and (B) Abz-AAPFSSKQ-EDDnp by SR5-3 protease at 37 °C in theabsence of salt (○), 3 M NaCl ( ) and 1 M Na2SO4 (●). Ratios of kcat/Km measured inmixed isotopic solvents over the kcat/Km measured in water were plotted against theatom fraction of deuterium, n, and curves have been calculated from best-fit modelsoutlined in Table 1.

370 D.N. Okamoto et al. / Biochimica et Biophysica Acta 1794 (2009) 367–373

3.2. Effects of salts on the kinetic parameters

The activation of protease SR5-3 by kosmotropic (1 M Na2SO4)and neutral (3 M of NaCl) salts was also investigated by theevaluation of the kinetic parameters (kcat, Km and kcat/Km) for thehydrolysis of the peptides Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp. Both contain the same non-prime site sequence but Abz-AAPFSSKQ-EDDnp includes the prime site sequence of SSKQ-EDDnpinstead of MCA. As shown in Table 2, the increase of kcat/Km valuesfor Suc-AAPF-MCA hydrolysis by SR5-3 in the presence of NaCl (17-fold) or Na2SO4 (29-fold) primarily occurred due to an increase inkcat. In contrast, the salt activation factor (R in Table 2) of the kcat/Km

values for the hydrolysis of the FRET peptide Abz-AAPFSSKQ-EDDnpis significantly lower than observed for the hydrolysis of Suc-AAPF-MCA. It is noteworthy that in the absence of salt the peptide Suc-AAPF-MCA is hydrolyzed with kcat/Km value 33-fold lower than thatof Abz-AAPFSSKQ-EDDnp (associated with a 15-fold increase in theKm value). Therefore, for the hydrolysis of Abz-AAPFSSKQ-EDDnp thesubstrate–enzyme interactions at the prime subsites attenuate thefavorable effects of the salts observed on the hydrolysis of Suc-AAPF-MCA.

3.3. Proton inventory (PI) for the hydrolysis of Suc-AAPF-MCA andAbz-AAPFSSKQ-EDDnp

Deuterium kinetic solvent isotope effect on enzymatic reactionsis the best method to evaluate the nature of proton transfer ingeneral acid–base catalyzed reactions that promote formation anddecomposition of the tetrahedral intermediate involved in theacylation and in deacylation steps of the serine protease catalyticmechanism [20–21]. Proton inventory methodology is an extensionof the solvent isotope effect method in which the reactionparameter k is expressed as kn(n), a function of the deuterium

Table 2Kinetic parameters for hydrolysis by SR5-3 of fluorescent and FRET peptides in absence and

Peptides Absence of salt 3 M NaCl

kcat (s−1) Km (μM) kcat/Km (mM s)−1 kcat (s−1) Km (

Suc-AAPF-MCA 8.0±0.4 198±10 40±2 136.5±6.8 198±Abz-AAPF↓SSKQ-EDDnp 16.9±0.8 12.8±0.6 1321±66 8.0±0.4 2.3±

Conditions for hydrolysis: Assays were performed at 37 °C, in 50 mM Tris–HCl pH 8.0. Theabove and below the Km values. aR is the ratio of kcat/Km values in the presence and absence othree independent measurements. The hydrolyzed bonds (↓) were determined by HPLC and

atom fraction n presents in the isotopic solvent. The shape of theobtained function permits determination of the number of protonbridges in the rate-determining step of the reaction, and is adiagnostic tool of the reaction mechanism [21].

Normal kinetic solvent isotopic effects were observed for thehydrolysis of Suc-AAPF-MCA, with kinetic values in the range 1.77and 1.99. As shown in Fig. 3A, the increase of D2O in the kineticbuffer results in similar effects on kcat/Km values either in theabsence or presence of NaCl or Na2SO4. Table 3 shows thefractionation factors for the fitting of the data obtained with themodels (i.e. equations described in Table 1) used for the protoninventory study. The observed domed proton inventories areconsistent, in all cases, with two proton bridges at the TS plus asolvent reorganization that contributes with an inverse component.This data is in agreement with the formation of the acyl–enzymetetrahedral intermediate complex as the rate determining step forthis reaction, as usually described for serine-peptidases [20]. More

presence of NaCl or Na2SO4

1 M Na2SO4

μM) kcat/Km (mM s)−1 aR kcat (s−1) Km (μM) kcat/Km (mM s)−1 aR

10 689±34 17 149.5±7.5 129±6.4 1155±57.7 290.1 3485±174 2.6 4.8±0.24 0.95±0.05 5084±254 3.8

concentrations of the substrates employed in each determination were at least 5 timesf salt. The errors for determination of the kinetic parameters were obtained from at leastmass spectroscopy.

Table 3Fractionation factors fitting various models from Table 1 for the hydrolysis of Suc-Ala-Ala-Pro-Phe-MCA by SR5-3 in Tris 50 mM pH8.0a

Salt Ø1 Ø2 ØSolv X2

TS1 – 0.66±0.02 – – 0.002486TS1+Solv – 0.30±0.03 – 1.83±0.10 0.0003962TS1 – 0.82±0.02 – (1.00) 0.0028382TS1+Solv – 0.43±0.02 – 2.93±0.10 0.000285TS1+TS2 – 0.35±0.02 1.62±0.06 (1.00) 0.000297TS1+TS2+Solv – 0.45±1.87 0.42±1.89 2.93±0.25 0.0003322TS1+Solv NaCl 0.42±0.03 – 2.80±0.25 0.0004712TS1+Solv Na2SO4 0.46±0.04 – 2.61±0.38 0.000112

a Most consistent models are denoted in bold face characters.

Fig. 5. Effect of temperature on Km value of SR5-3 hydrolysis of Abz-AAPFSSKQ-EDDnpin absence of salt (○), 3 M NaCl ( ) and 1 M Na2SO4 (●). Assay conditions are describedin Materials and methods.

371D.N. Okamoto et al. / Biochimica et Biophysica Acta 1794 (2009) 367–373

important, this data clearly indicates that the salt activation doesnot change the number of protonic sites and their mode ofparticipation in the SR5-3 catalysis of non-prime-side substrates.

In contrast, as shown in Fig. 3B, for the hydrolysis of the peptideAbz-AAPFSSKQ-EDDnp by SR5-3 an inverse isotopic effect wasobserved in all assay conditions. This result suggests that D2Oproduces conformational changes (and solvent reorganization at theprime substrate sites) in SR5-3 that are detected only in the hydrolysisof substrates that occupy the prime subsites of the enzyme. In thepresence of NaCl or Na2SO4 the effects of D2O are reduced, indicatingthat salts increased the enzyme rigidity.

3.4. Temperature dependence of SR5-3 activity

Fig. 4 shows the Eyring plot for the kcat/Km values of thehydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3protease in the absence of salt or in the presence of 3 M NaCl or 1 MNa2SO4 over the temperature range 10–55 °C. SR5-3 was stable inthe studied temperature range; after incubation at 55 °C the enzyme

Fig. 4. Eyring plot for the hydrolysis of (A) Suc-AAPF-MCA and (B) Abz-AAPFSSKQ-EDDnptemperature range 10–55 °C. Assay conditions are described in Materials and methods.

gives the same rate constant at 37 °C as measured without pre-incubation. Except for the hydrolysis of Abz-AAPFSSKQ-EDDnp inthe absence of salt, convex Eyring plots were observed in all otherassay conditions. In the presence of salts, deviation of the plotsstarts at a significantly lower temperature (∼25 °C) for thehydrolysis of Abz-AAPFSSKQ-EDDnp compared to the hydrolysis ofSuc-AAPF-MCA (∼45 °C). The differences are even more evident athigher temperatures where the slope related to ΔH⁎ becomes lessnegative for Suc-AAPF-MCA hydrolysis, and the negative ΔH⁎ turnedto positive for Abz-AAPFSSKQ-EDDnp hydrolysis. It should bepointed out that in the hydrolysis of Abz-AAPFSSKQ-EDDnp thisreduction in the specificity constant is related to a 10-fold increase

by SR5-3 protease in absence of salt (○), 3 M NaCl ( ) and 1 M Na2SO4 (●) over the

Table 4Activation parameters for the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnpby SR5-3 protease

ΔH⁎ (KJ/mol) ΔS⁎ (J/mol/K) ΔG⁎ (KJ/mol)

Suc-AAPF-MCAAbsence of salt 47 4 463 M NaCl 41 7 391 M Na2SO4 31 −27 39

Abz-AAPFSSKQ-EDDnpAbsence of salt 30 −30 393 M NaCl 55 64 361 M Na2SO4 41 21 35

Assay conditions as described in Materials and methods.

372 D.N. Okamoto et al. / Biochimica et Biophysica Acta 1794 (2009) 367–373

in Km above 25 °C in the presence of salts compared to 1.7-fold in itsabsence (Fig. 5). These results are in agreement with a change in therate-limiting step as temperature is increased, because for thegeneral mechanism of serine-peptidases, the kcat/Km reflects theformation of Michaelis complex at lower temperatures (where k2-bk−1), and would be affected by substrate dissociation from theactive site at higher temperatures (where k−1bbk2 and kcat/Km=k1k2/k−1 [29]).

The linear Eyring plot at lower temperatures allowed the calcula-tion of entropy (ΔS⁎), enthalpy (ΔH⁎), and Gibbs energy of activation(ΔG⁎) associated with the formation of the ES complex. The largestdifferences for the hydrolysis of Suc-AAPF-MCA compared to that ofAbz-AAPFSSKQ-EDDnp were observed in the ΔS⁎ values (Table 4).Although a negative entropic contribution is expected to result fromthe freezing in translational and rotational motion that occurs whenthe reactants go from the ground state (E+S) to the enzyme–substratecomplex (ES), solvent effects interferewith the entropy change and canaccount for its positive value [30,31].

4. Discussion

Subtilisin-like serine proteases (subtilases) are widely distributedin various organisms, including bacteria, archaea, and eucaryotes [32].The structure and function of the subtilisin Carlsberg from Bacilluslicheniformis and subtilisin BPN from B. amyloliquefaciens [33–34]have been extensively studied and pointed out to have a catalyticmechanism similar to chymotrypsin-like serine proteases [20,35].However, despite the economic interest associated with biotechnol-ogy applications, little is known about the kinetic mechanism of saltactivation on halophilic serine-proteases, particularly of halophilicsubtilases.

We observed that catalytic activity of SR5-3 protease is enhancednot only by NaCl but also by kosmotropic salts of the Hofmeisterseries, including sodium citrate, Na2SO4, and Na2HPO4. These saltsare preferentially excluded from the solvation shell of proteins dueto their higher affinity for water than for the protein surface. As aconsequence, the tendency of the protein is to change itsconformation, reducing the exposure of its surface by a salting outmechanism [11,13,36–38].

It is noteworthy that the salt modulation of SR5-3 peptidaseactivity was dependent on the occupancy of its prime site subsitesby the substrate, as demonstrated by the kinetic parameters for thehydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp (Table 2).The hypothesis that salt could elicit the full catalytic power of SR5-3halophilic protease inducing multi-proton catalysis and beingresponsible for the increase of nucleophilic attack and directlyaffecting kcat was investigated by solvent isotope effect analysis andby proton inventory profiles. In agreement with the general basecatalysis mechanism proposed for serine proteases, normal solventisotope effects and domed proton inventories were observed in allassay conditions for the SR5-3 hydrolysis of Suc-AAPF-MCA,

indicating that two proton bridges are formed at the TS and thatthere is a significant solvent reorganization associated with thisprocess, either in presence or absence of salts. This result refutes thehypothesis that the catalysis involves high numbers of protonbridges at the transition state formation. Moreover, the occurrenceof an inverse isotope effect for the hydrolysis of Abz-AAPFSSKQ-EDDnp suggests that the rate limiting step of both peptides aredifferent, with the second being related to a protease conformationrather than a chemical step.

A different mechanism that can be involved in salt activation ofsmall peptides is catalysis by distortion [30]. According to thismechanism, nucleophilic attack of the active site serine hydroxylgroup on the carbonyl carbon of the amide substrate can beimproved if this bond is twisted out of its planar stableconfiguration. This distortion disrupts the resonance stabilizationof the amide and renders the carbonyl moiety of the amide moreester-like, and thus a more reactive configuration toward nucleo-philic attack. In this model, if salt promotes the isomerization ofenzyme to a configuration that induces the distortion mechanism,this effect would be more limited in the hydrolysis of the peptideAbz-AAPFSSKQ-EDDnp compared to that of Suc-AAPF-MCA. Thiscould be due to the Michaelis complex stabilization provided by theoccupancy of S1′ to S4′ enzyme subsites by the substrate Abz-AAPFSSKQ-EDDnp. The effect of these prime site interactions canalso be addressed by the thermodynamic values of ES activation(Table 4), particularly the large variations of ΔS⁎ values in thepresence of salts, indicating that they improve the enzyme–substrate interactions in the Michaelis complex.

In conclusion, our results support the notion that the salting outeffect is responsible for the halophilic character of SR5-3. Themagnitude of the activation of the peptide substrate hydrolysis ismainly derived from the improvement of catalytic and/or interactionsteps depending on whether substrate occupies the enzyme primesubsites.

Acknowledgments

This work was supported by the Brazilian research agenciesFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)and Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq). The authors thank Prof Michael Blaber for editing the Englishtext.

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