Engineering the xenobiotic substrate specificity of maize glutathione S-transferase I

Nikolaos E. Labrou1, Georgia A. Kotzia and Yannis D. Clonis

Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, 11855 Athens, Greece

1 To whom correspondence should be addressed. E-mail: lambrou{at}aua.gr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Glutathione S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of glutathione (GSH) to electrophilic sites on a variety of hydrophobic substrates. In the present study three amino acid residues (Trp12, Phe35 and Ile118) of the xenobiotic binding site (H-site) of maize GST I were altered in order to evaluate their contribution to substrate binding and catalysis. These residues are not conserved and hence may affect substrate specificity and/or product dissociation. The results demonstrate that these residues are important structural moieties that modulate an enzyme's catalytic efficiency and specificity. Phe35 and Ile118 also participate in kcat regulation by affecting the rate-limiting step of the catalytic reaction. The effect of temperature on the catalytic activity of the wild-type and mutant enzymes was also investigated. Biphasic Arrhenius and Eyring plots for the wild-type enzyme showed an apparent transition temperature at 35°C, which seems to be the result of a change in the rate-limiting step of the catalytic reaction. Thermodynamic analysis of the activity data showed that the activation energy increases at low temperatures, whereas the entropy change seems to be the main determinant that contributes to the rate-limiting step at high temperatures.

Keywords: glutathione S-transferase/herbicide detoxification/protein engineering/xenobiotic substrate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The glutathione S-transferases (GSTs, EC 2.5.1.18) comprise a large family of ubiquitous detoxifying enzymes. GST catalyses the nucleophilic attack of the sulphur atom of the tripeptide glutathione (GSH) on electrophilic groups of a variety of hydrophobic substrates (Sheehan et al., 2001Go). In plants, GSTs are grouped into five classes based on their amino acid sequences, namely theta, zeta, phi, tau and omega. Whereas zeta, theta and omega classes of GSTs are found in both plants and animals, the phi and tau classes are unique to plants (Dixon et al., 1998Go; Edwards et al., 2000Go). Each soluble GST is a dimer of ~26–29 kDa subunits. Each subunit contains an independent catalytic site composed of two components (Armstrong, 1998Go; Sheehan et al., 2001Go). The first is a binding site specific for GSH (the G-site), formed from a conserved group of residues located in the N-terminal domain of the polypeptide. The second component is a site that binds a variety of hydrophobic substrates (the H-site), which is much more structurally variable and is formed from residues located in the C-terminal domain. The structural biology of GSTs derived from different enzyme classes has been studied in detail. High-resolution crystal structures are available for phi GSTs from Arabidopsis (Reinemer et al., 1996Go) and maize (Neuefeind et al., 1997aGo,bGo; Prade et al., 1998Go) and for tau (Thom et al., 2002Go) and zeta (Thom et al., 2001Go) classes from wheat and Arabidopsis, respectively. Despite the high sequence divergence between the GST classes, the overall structures of the enzymes are remarkably similar.

Detailed studies of GSTs are justified because of the considerable agronomic and therapeutic potential of the enzyme in, for example, the development of transgenic plants with increased resistance to biotic and abiotic stress (Roxas et al., 1997Go; Milligan et al., 2001Go; Dixon et al., 2003Go) and the design of anticancer gene therapy drugs for protecting normal cells from chemotherapeutics (Yue et al., 2003Go).

The isozyme GST I from maize has been the major focus of interest as enzyme model for herbicide detoxification (McGonigle et al., 2000Go; Labrou et al., 2001aGo,bGo, 2004Go). It is the most abundant GST isoenzyme in maize (McGonigle et al., 2000Go), showing constitutive expression in seedlings and forming a homodimer protein of 214 amino acids.

In this paper,we address questions regarding the role of selected residues located in the H-site, with regard to the catalytic function and substrate specificity. The results of the present work form the basis for a rational design of new engineered GSTs with altered specificity and enhanced catalytic efficiency towards different herbicides. Furthermore, they may help in the design of new, more selective and environmentally friendly herbicides.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
dNTPs and restriction enzymes were obtained from Roche (Nutley, NJ), reduced glutathione, ethacrynic acid and CDNB from Sigma (St Louis, MO) and Pfu DNA polymerase and all other molecular biology reagents from Promega (Madison, WI).

Cloning, expression and purification of maize GST I

Cloning of maize GST I into a pQE70 expression vector to yield the pQEGST expression plasmid was described by Labrou et al. (2001b)Go. Expression and purification of GST I were performed according to Labrou et al. (2001b)Go.

Kinetic analysis

Enzyme assays for the CDNB conjugation reactions were performed at 30°C (unless otherwise stated) according to a published method (Labrou et al., 2001bGo). Steady-state kinetic measurements were performed in 0.1 M potassium phosphate buffer, pH 6.5. Initial velocities were determined in the presence of 2.5 mM GSH and the CDNB concentration was varied in the range 0.05–1 mM. Alternatively, CDNB was used at a fixed concentration of 1 mM and the glutathione concentration was varied in the range 0.2–2 mM. Enzyme assays for the ethacrynic acid conjugation reactions were performed at 30°C according to a published method (Micaloni et al., 2000Go). Solutions of glutathione were prepared fresh each day and stored on ice under N2. All initial velocities were determined in triplicate. The kinetic parameters kcat and Km were calculated by non-linear regression analysis of experimental steady-state data using the GraFit (Erithacus Software) program (Leatherbarrow, 1998Go).

Site-directed mutagenesis

Site-directed mutagenesis was performed according to Weiner et al. (1994)Go. The pairs of oligonucleotide primers used in the PCR reactions were as follows: for the Ile118Phe mutation, 5'-CCCATCAACCTCGCCACCGCC-3' and 5'-CACGATCTCGTAGTCGGAGCC-3'; for the Trp12Pro mutation, 5'-ATGTCGCCGAACGTGACGAGGTGC-3' and 5'-CACCGCCCCGTACAGCTTCATCGG-3'; for the Trp12Ile mutation, 5'-ATGTCGATCAACGTGACGAGGTGC-3' and 5'-CACCGCCCCGTACAGCTTCATCGG-3'; and for the Phe35Leu mutation, 5'-ATCAACCTCGCCACCGCCGAGCAC-3' and 5'-GGGCACGATCTCGTAGTCGGAGCC-3'. The expression construct pGST I encoding maize GST I was used as template DNA in all mutagenesis reactions. All mutations were verified by DNA sequencing. Expression and purification of mutated forms of GST I were performed as described for the wild-type enzyme (Labrou et al., 2001bGo; Axarli et al., 2004Go).

Viscosity dependence of kinetic parameters

The effect of viscosity on kcat was assayed at 30°C, in 0.1 M potassium phosphate buffer, pH 6.5, containing various glycerol concentrations. Viscosity values ({eta}) were calculated as described by Wolf et al. (1985)Go. Glycerol does not induce changes in the enzyme secondary structure as detected by far-UV difference spectroscopy. Furthermore, glycerol does not have any inhibitory effect on catalysis (Labrou et al., 2001aGo).

Effect of temperature on enzyme activity

The dependence of the reaction rate on temperature was evaluated by measuring kcat at different temperatures (5–60°C) under the same conditions as reported above. The data were analysed by Arrhenius and Eyring plots as described in the Results and discussion section. In cases where the data were non-linear, the linear portions were fitted to the Arrhenius equation separately.

Far-ultraviolet difference spectra were measured with a Perkin-Elmer Lamda 16 recording spectrophotometer at different temperatures (25–60°C). The enzyme (0.05 mg/ml) was dialysed against 0.01 M potassium phosphate buffer, pH 6.5, and its ultraviolet spectrum was recorded between 190 and 220 nm against enzyme solution at 25°C (Labrou et al., 2004Go).

Spectroscopic studies

Difference spectra of GSH bound to the wild-type and mutant enzymes were obtained with a Perkin-Elmer Lamda 16 double-beam double monochromator UV–VIS spectrophotometer equipped with a cuvette holder that was thermostated at 25°C as described by Labrou et al. (2001a)Go. In a typical experiment, 1 mM GSH (final concentration) was added to GST I (~10 µM active sites) in 1 ml of 0.1 M suitable buffer. The amount of thiolate formed at each pH was monitored with the peak-to-trough amplitude between 240 and 300 nm on the basis of an {varepsilon}240 nm of 5000 M–1 cm–1 after subtraction of the spectral contributions of free enzyme and of free GSH, dissolved in BSA solution showing an A240 similar to that of the GST I sample. pKa values were obtained by fitting the data to the equation

(A)

Bioinformatics and molecular modelling

Sequences homologous to GST I were sought in the NCBI using BLAST (Altschul et al., 1990Go) and PSI-BLAST (Altschul et al., 1997Go). The resulting sequence set was aligned with Clustal W (Higgins et al., 1994Go). ESPript (http://prodes.toulouse.inra.fr/ESPript/cgi-bin/ESPript.cgi) was used for alignment visualization and manipulation.

Mutations modelling and computations were done with the GROMOS96 implementation of Swiss-PdbViewer. Calculations were done in vacuo with the GROMOS96 43B1 parameters set, without reaction field. A model of the isoenzyme GST IV from maize (NCBI accession number X79515) was constructed using MODELLER 6 (Sali and Blundell, 1993Go; run at http://www.infobiosud.cnrs.fr/bioserver). The determined X-ray crystal structure of maize GST I, with which GST IV shares 56% sequence identity, was used as a template. The structures of the GST I in complex with lactoyl-GSH and atrazine-GSH conjugates have been determined at 2.5 Å (PDB code 1axd; Neuefeind et al., 1997aGo) and 2.8 Å (PDB code 1bye; Prade et al., 1998Go) resolutions, respectively. We chose to use the 1bye structure, despite its slight poorer resolution, because we wished to model the ligand-bound state of GST IV and the 1bye structure was more informative. An alignment of the GST I and GST IV was generated with Clustal W and analysed by TITO (Labesse and Mornon, 1998Go). An iterated protocol involving multiple model construction (four for each tested alignment) and rigorous protein structure quality assessment, using PROSA II (Sippl, 1993Go) and Verify 3D (Luthy et al., 1992Go), was used. PROSA II and Verify 3D both yield overall scores and also local profiles which can be used to localise areas of unusual packing and/or solvent exposure characteristics. The overall scores were used to choose the final model. Analysis of packing, solvent exposure and stereochemical properties suggests the final GST IV model to be of good overall quality.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Selection of residues for mutagenesis

The isoenzyme GST I from maize has been the subject of intense research because of its ability to detoxify a wide range of xenobiotics. It belongs to class phi and to type I of GSTs, according to the classifications of Edwards et al. (2000)Go and McGonigle et al. (2000)Go, respectively. On the basis of the available X-ray crystal structure of GST I (Neuefeind et al., 1997aGo; Prade et al., 1998Go), three residues (Trp12, Phe35 and Ile118; Figure 1A) of the H-site were selected for assessing their contribution to substrate binding and catalysis. The H-site of GST I exhibits a unique structure and is built predominantly of hydrophobic residues from helix H'''3, (Ile118), the N-terminus (Trp12) and helix H2 (Phe35). These residues are not conserved among class I GSTs (Figure 2) and have been proposed to modulate substrate recognition by affecting the size and the shape of the H-site. These residues were individually mutated to the equivalent residues found in phi class maize GST III or Arabidopsis GST (AraGST). In particular, Trp12 was mutated to Pro and Ile, Phe35 was mutated to Leu and Ile118 was mutated to Phe. The mutation of these catalytic residues is believed to help understand better the factors that affect substrate specificity of phi class GSTs.



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Fig. 1. Structural representation depicting important residues of maize GST I. (A) Bound S-atrazine-glutathione (dark grey) conjugate is shown in a stick representation. Trp12, Phe35 and Ile118 are drawn in a stick representation and are shown in light grey. (B) The proposed sandwich complex formed between Phe35, the aromatic ring of the substrate and Phe118 in the mutant Ile118Phe.

 


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Fig. 2. Sequence alignments of type I maize GSTs with A.thaliana PM24. The alignments were produced using ESPript. The secondary structure of GST I, and GST I numbering are shown above the alignment. {alpha} helices and ß strands are represented as helices and arrows, respectively, and ß turns are marked with TT. NCBI accession numbers for maize type I GSTs are in parentheses: GST I (M16901); GSTIII (X04455); GSTIV (X79515); GST8 (AF244673); GST9 (AF244674); GST10 (AF244675); GST12, (AF244677); GST13 (AF244678); GST14 (AF244679); GST15 (AF244680). The accession number for A.thaliana GST is P46422.

 
Effect of point mutations on Km for CDNB and GSH

The kinetic parameters kcat and Km for the wild-type and mutant enzymes (Trp12Ile, Trp12Pro, Phe35Leu, Ile118Phe) were determined by steady-state kinetic analysis and the results are given in Table I. All mutants show differences in Km values for CDNB compared with the wild-type enzyme. In particular, mutant Phe35Leu and Trp12Ile showed an increase of Km of about 13-fold and 4.3, respectively, whereas mutant Ile118Phe shows a decrease of about 8-fold, suggesting higher affinity for CDNB. In addition, mutant Phe35Leu showed an increase in Km value for GSH. These findings may be interpreted on the basis of the X-ray structure of GST I. Phe35 is located in the flexible segment of residues 35–45 and is involved in a {pi}{pi} aromatic interaction with the xenobiotic substrate. In the same segment are also located the residues His40 and Lys41, which form salt bridges with the Gly carboxylate of GSH. The Phe35Leu mutation may have two semi-independent consequences: the loss of a {pi}{pi} aromatic interaction of the Phe side chain with the xenobiotic substrate and the introduction of a cavity. The cavity could either be filled by new water molecules or by a rearranged local protein structure. Either the loss of these interactions or the rearrangement of the loop could affect His40 and Lys41 conformations. In addition, Phe35 is involved in direct amino-aromatic interaction with His40. Amino-aromatic interaction has been found in several proteins to have certain structural and mechanistic implications such as the increase of stabilising energy and the elevation of the pKa value of the His (Shoemaker et al., 1990Go; Loewenthal et al., 1992Go). All these consequences would affect CDNB and GSH binding and would result in reduced affinity (increased Km) of the enzyme for both substrates. On the other hand, the Ile118Phe mutation may have as a consequence the development of a new {pi}{pi} aromatic interaction with the aromatic ring of the substrate. Computer modelling shows that the ring of the aromatic substrate may be packed between the two aromatic rings of Phe35 and Phe118 (Figure 1B). The resulting sandwich complex may explain the increased affinity of the mutant for CDNB. A similar complex has also been observed in the pi class GST from human placenta which exhibits high affinity for aromatic substrates (Oakley et al., 1997Go).


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Table I. Steady-state kinetic parameters of wild-type and mutants of maize GST I for the CDNB conjugation reaction

 
Trp12 is involved in a weak hydrophobic interaction with the aromatic ring of the xenobiotic substrate. In the mutants, the smaller side chain of Pro or Ile provides more room, compared with that of Trp side chain, which may lead to unproductive binding of substrate. Interestingly, the mutant Trp12Pro shows a decreased Km value for GSH. This unexpected finding may be due to the indirect effect of Trp12Pro mutation on the architecture of GSH binding residues. Computer modelling of the Trp12Pro-mutated enzyme, followed by energy minimization, shows that some of the GST binding residues, such as Ser67 and Arg68, adopt more favourable interactions with the GSH molecule. For example, Ser67, which is involved in direct interaction with the {gamma}-Glu-carboxylate moiety of GSH, in the mutated enzyme exhibits about 25% higher non-bonded and electrostatic energy, as calculated after energy minimization (GROMOS 96 software, 43B1 parameters set). This finding may further be confirmed by the observation that the Km for GSH of the isoenzyme GST IV from maize, which also has Pro at position 12, is 0.29 mM (Figure 2) (Irzyk and Fuerst, 1993Go) and the Km of the isoenzyme AraGST from Arabidopsis, which has Ile at this position, is 0.08 mM (Bartling et al., 1993Go). The above finding further supports our observation of the indirect role of the residue at position 12 in the formation of the G-site.

Effect of point mutations on kcat for the CDNB conjugation reaction

All mutants showed a decrease in kcat value (Table I). The contribution of the mutated residues to kcat may be due to their involvement either in regulating the transition-state structure or in the rate-limiting step of the catalytic reaction. Therefore, the effect of viscosity on kcat was measured for the mutant and for the wild-type enzyme. A decrease in kcat by increasing the viscosity of the medium should indicate that the rate-limiting step of the reaction is related to the product release or to diffusion-controlled structural transitions of the protein (Sampson and Knowles, 1992Go; Johnson et al., 1993Go). A plot of the inverse relative rate constant kcat°/kcat (kcat° is determined at viscosity {eta}°) versus the relative viscosity {eta}/{eta}° should be linear with a slope equal to unity when the product release is limited by a strictly diffusional barrier or a slope approaching zero if diffusion-controlled structural transitions of the protein are rate-limiting (Sampson and Knowles, 1992Go; Johnson et al., 1993Go). The inverse relative rate constant kcat°/kcat, determined at 30°C, for the wild-type enzyme-catalysed reaction shows a linear dependence on the relative viscosity with a slope of 1.1 ± 0.1, suggesting that the product release is rate-limiting for the wild-type enzyme (Figure 3) (Labrou et al., 2001aGo,bGo). Diffusion-controlled phenomena of the protein have already been reported to modulate the catalysis of other GST isozymes: a conformational change in the case of the ternary complex of human P1-1 (Caccuri et al., 1996Go) and product release for rat GST M1-1 (Johnson et al., 1993Go), Lucilia cuprina GST (Caccuri et al., 1997Go) and housefly I1 (Nay et al., 1999Go).



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Fig. 3. The effect of viscosity on kinetic parameters at pH 6.5 and 30°C. Plot of the reciprocal of the relative turnover number (kcat°/kcat) as a function of relative viscosity ({eta}/{eta}°) with glycerol as cosolvent for the wild-type (open diamonds), and for the mutants Phe35Leu (closed squares) and Ile118Phe (closed diamonds). Experiments were performed in triplicate and lines were calculated by least-squares regression analysis.

 
Mutants Phe35Leu and Leu118Phe showed different degrees of dependence on viscosity, compared with the wild-type enzyme, indicating that structural transitions contribute to the rate-limiting step of these enzyme variants. On the other hand, the mutant Trp12Ile showed no appreciable difference compared with the wild-type enzyme (data not shown). Taking into account the involvement of Phe35 in the induced-fit mechanism of Helix2 (Neuefeind et al., 1997aGo,bGo) and of Ile118 in the structural organization of Helix3, it seems plausible that the enzyme's rate-determining structural fluctuations may involve changes in conformation/dynamics of Helix2 and Helix3.

Another possible factor that may contribute to the reduction of kcat in the mutated enzymes is a shift in the pKa of the reactive thiol group of GSH. The crucial property of GSTs is their ability to lower the pKa of the thiol group of the bound GSH (Caccuri et al., 1997Go; Armstrong, 1998Go; Labrou et al., 2001aGo). Direct demonstration of the formation of GSH thiolate at the active site and its dependence on pH for the wild-type enzyme has already been studied by difference spectroscopy (Labrou et al., 2001aGo). This study revealed that the difference spectrum of the binary complex GST I–GSH, shows an absorption band centred at 240 nm, that is diagnostic of a thiolate anion (Labrou et al., 2001aGo). The pH dependence of the absorption band at 240 nm identified an apparent pKa of 6.2 for the bound thiol ionization for the wild-type enzyme (Labrou et al., 2001aGo). In order to investigate the influence of the H-site residues Trp12, Phe35, Ile118 on GSH thiol ionization, difference spectroscopy experiments were also performed for the mutated enzyme forms (Table I). The results showed that the mutations did not seem to significantly change the pKa of bound GSH. This finding was expected, since the side chains of the mutated residues are located away (~6 Å) from the SH group of GSH and do not exhibit acid-base properties.

Kinetic analysis of the effect of replacement of H-site residues on ethacrynic acid conjugation reactions

To confirm further the data obtained by investigating the CDNB conjugation reaction, kinetic analysis of the wild-type and mutants was carried out to evaluate the effect of each mutation on the catalytic parameters of the ethacrynic acid (EA) conjugation reaction. EA is a known inhibitor of GSTs but in addition can act as a substrate for maize GST I (McGonigle et al., 2000Go). EA is thought to form a conjugate with GSH via Michael addition, both spontaneously and by GST-driven catalysis. The {alpha},ß-unsaturated ketone moiety is the target for conjugation by GSH. The kinetic parameters Km and kcat, as determined by steady-state kinetic analysis, are listed in Table II. From the data presented in Tables I and II, it is evident that, although in absolute terms the kinetic constants differ significantly, in relative terms the kinetic constants follow some general trends. This suggests that the proposed amino acid residues also contribute to the EA conjugation reaction and establish their important role in substrate binding and catalysis. It should be noted that a well known property of GSTs is that different amino acids contribute to different substrates (Prade et al., 1998Go).


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Table II. Steady-state kinetic parameters of wild-type and mutants of maize GST I for the ethacrynic acid conjugation reaction

 
Effect of temperature on the activity of the wild-type and mutant enzymes

To study the effect of temperature on kcat of the CDNB conjugation reaction, the activity data were analysed by plotting the logarithm of activity versus the reciprocal of the absolute temperature and fitted to the Arrhenius equation (Equation 1) (Oakes et al., 2003Go; Peng et al., 2003Go):

(1)
where Ea is the activation energy, R is the gas constant and Z is the pre-exponential factor. The results are given in Tables III and IV for the wild-type and mutants, respectively.


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Table III. Apparent activation energies and activation entropies of the reaction of the wild-type GST I

 

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Table IV. Apparent activation energies and activation entropies of the reaction of the mutants of GST I

 
The activity of GST I increased with increasing temperature. A fitting of experimental data to the Arrhenius equation shows the presence of two slopes for the wild-type, but one slope for the mutants (Figure 4). The activation energies are much higher at low than at high temperatures (Table III). It should be emphasized that the decline in the curve with increase in temperature was not due to denaturation. All mutants exhibit higher activation energies than the wild-type enzyme (Table III). Mutants Trp12Pro and Trp12Ile exhibit the highest increase of the activation energy and, therefore, Trp12 seems to be the main contributor to the transition state formation. This conclusion is further supported by the calculated values that will be discussed below.



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Fig. 4. Effects of temperature on GST I activity. The data were fitted to the Arrhenius equation for the wild-type (closed square), and for the mutants Phe35Leu (open squares), Ile118Phe (five-pointed stars), Trp12Ile (asterisks) and Trp12Pro (open diamonds). In the wild-type enzyme, where the data were non-linear, the linear portions at low or high temperatures were fitted to the Arrhenius equation separately. The y-axis is in arbitrary units to facilitate comparison of the different lines.

 
Non-linear Arrhenius plots may be attributed either to a change in the rate-limiting step of the reaction or to a conformation change of the enzyme (Oakes et al., 2003Go). To test the possibility that the transition observed in the Arrhenius plot for the wild-type enzyme has a conformational origin, we investigated the temperature dependence of the far-UV spectra of the enzyme (Figure 5). This can serve as indicator of the conformational state of the protein (Rosenheck and Doty, 1961Go). Changes in protein conformation are evident from the difference spectra (data not shown). The perturbations observed in the spectra at 200 nm are due to changes in the secondary structure of the protein (Rosenheck and Doty, 1961Go; Kotzia and Labrou, 2004Go). These conformational changes in the secondary structure of GST I exhibit a temperature dependence with an inflection point at 55°C (Figure 5), which is different from the temperature at which the transition of the Arrhenius plot was observed (35°C). Therefore, the results suggest that the non-linear Arrhenius plot is probably caused by a change in the rate-limiting step of the catalysis. However, a small change that appeared between 30 and 40°C (Figure 5) may affect the catalytic efficiency without evident changes in the secondary structure and therefore we cannot rule out that conformational changes may contribute, to some extent, to the biphasic Arrhenius plot. The negative entropy change associated with the change in the rate-limiting step for the wild-type enzyme suggests that the new rate-limiting step results from possible less tight hydrophobic interactions (Table III).



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Fig. 5. Temperature dependence of far-UV difference spectra of GST I in potassium phosphate buffer (0.01 M) pH 6.5. The plot depicts the dependence of the absorption at 200 nm versus temperature.

 
To analyse further the effect of point mutation on the transition state formation, the activity data shown in Figure 5 were fitted to the Eyring equation (Equation 2) and to a modified form of the Eyring equation (Equation 3) (Fan et al., 2000Go). According to Eyring's transition state theory, the temperature dependence of kcat is given by Equation 2 (Fan et al., 2000Go, Kong et al., 2003Go):

(2)
where kß is Boltzmann's constant, h is Planck's constant, R is the gas constant and {Delta}G!=, {Delta}H!= and {Delta}S!= are the free energy, enthalpy and entropy of activation of the rate-limiting step in the reaction, respectively. The data are plotted as the logarithm of kcat/T versus the reciprocal of the absolute temperature and fitted to Equation 3. The results are shown in Tables III and IV for the wild-type and mutants, respectively. A positive {Delta}H!= was expected because the transition state involves the breaking of bonds.

(3)
where k°cat is the rate constant at T0 (T0 = 25°C = 298.15 K), is the temperature-dependent enthalpy of activation and is the temperature-independent change in specific heat at an arbitrary reference temperature. Temperatures that lead to non-linear Arrhenius plots also lead to non-linear Eyring plots with derived values of less than 1 kJ/mol·K. The derived changes in specific heat are within the range expected for processes involving proteins (Fan et al., 2000Go; Kong et al., 2003Go). For the mutants Trp12Pro and Trp12Ile we observed large differences in compared with the wild-type enzyme (Tables III and IV). Such differences in specific heat can result from changes in hydration state and may originate from releasing water molecules (Ortiz-Salmerón et al., 2003Go). This shows that the binding of substrates produces a higher degree of water molecule loss in the wild-type than that in the mutants and suggests that there are differences in the transition state structure.

Structural basis of substrate specificity

Within the maize GST isoenzymes there are significant differences in their individual activities for a wide variety of hydrophobic substrates (McGonigle et al., 2000Go). Comparison of the present data with those available in the literature show that the difference between the most active GST I and the least active GST IV is at least 3000-fold (McGonigle et al., 2000Go). Hence these proteins provide a unique opportunity for detailed comparison of how specific amino acids contribute to GST substrate specificity. To analyse the specificity of these isoenzymes, a structural model of GST IV was constructed and is shown in Figure 6. From the comparison of the structures of GST I and GST IV, several features that may contribute to substrate specificity can be discussed. GST IV exhibits about 70-fold lower activity for the herbicide alachlor and about 3000-fold lower activity for CDNB, compared with GST I. In this case the residue at position 35 is a positively charged Arg and the residues at position 12 and 118 are Pro and Phe, respectively (Figures 2 and 6). Exactly the same activity profile, as for GST IV, is seen in GST 8, which also has Arg residue at position 35, and the residues at position 12 and 118 are Pro and Ile, respectively. Arg does not provide the necessary hydrophobic environment for productive binding of hydrophobic substrates whereas the role of Pro at position 12 has already been discussed. In addition, the GST IV isoenzyme has high activity for the more hydrophilic ethacrynic acid. In this case Arg35 seems to provide the necessary positive charge, compared with GST I, for accommodating the negatively charged carboxyl group of ethacrynic acid. High activity for ethacrynic is also observed for the GST 8 isoenzyme, whereas GST10 with Met and Pro at positions 12 and 35 exhibits about 62% lower activity than GST IV and GST 8 towards ethacrynic acid (McGonigle et al., 2000Go).



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Fig. 6. Representation of the putative interactions of the atrazine-GSH conjugate with the GST IV model. The model was constructed as described in Materials and methods. The atrazine-GSH conjugate (dark grey) is drawn in a stick representation. Important residues (Arg35, Phe118 and Pro12) are drawn in a stick representation.

 
In conclusion, in this paper we have dealt with issues regarding the functional and structural roles of selected residues of the H-site of GST I, employing site-directed mutagenesis. From the practical standpoint, the diversity of GSTs and the differences in specificities may help towards the development of selective herbicides and drugs for future agricultural and medicinal use.


    Acknowledgments
 
This work was partially supported by the Hellenic General Secretariat for Research and Technology (programme: Operational Programme for Competitiveness, grant No. YB45).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received September 2, 2004; revised November 2, 2004; accepted November 3, 2004.

Edited by Vadim Mesyanzhinov





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