1 Chemistry Department, M.V. Lomonosov Moscow State University, 119899 Moscow, 3 A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov Str., 119991 Moscow, Russia and 4 Albany Molecular Research Inc., Biocatalysis Division, 2660 Crosspark Road, Coralville, IA 52242, USA
2 To whom correspondence should be addressed. E-mail: helena_koudriachova{at}hotmail.com
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Abstract |
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Keywords: -chymotrypsin/enzymeoligoamine complexes/stabilization/water/organic mixtures
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Introduction |
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To gain more insight into the key mechanisms that define the activation and stabilization effects of complex formation, information on the thermodynamic stability and stoichiometric composition of the complexes is needed. Enzymepolyelectrolyte complexes have been extensively studied during the last decade. However, thermodynamic parameters of the complexes were not determined since the composition and structure of the complexes depended significantly on the enzyme/polyelectrolyte ratio (Kudryashova et al., 2001). In this paper we propose that the use of shorter oligoelectrolytes instead of polyelectrolytes would help in estimating the thermodynamic parameters of the complexes formed due to electrostatic interactions.
In this study we investigated the complex formation of CT with a set of natural oligoamines (OA) having different chain lengths, spermine, spermidine and putrescine:
putrescine (put) NH2(CH2)4NH2
spermidine (smd) NH2(CH2)3NH(CH2)4NH2
spermine (sp) NH2(CH2)3NH(CH2)4NH(CH2)3NH2
One of the numerous biological functions of OA is to modulate the catalytic activity and stability of several enzymes (Tabor and Tabor, 1984). OA can be considered as polyelectrolytes (PE) with a low degree of polymerization. Like PE, OA are capable of forming electrostatic complexes with proteins (Tabor and Tabor, 1984
; Hashimoto et al., 1984
; Navaratnam et al., 1986
; Morozova et al., 1993
). The possibility of maintaining a catalytically active conformation by complex formation with OA has been demonstrated by examples of enhancing the thermal stability of complexed
-lactalbumin (Morozova et al., 1993
) and by stabilization of the enzyme against inactivation in waterorganic solvent systems (Kudryashova et al., 1999
). In biocatalytic applications the advantage of enzymeOA over enzymePE complexes (which were studied in much detail) is the possibility of performing the enzymatic reactions with high molecular mass substrates. Hence it is of great importance from the both fundamental and practical points of view to study the mechanisms of the formation and thermodynamic stability of the complexes of natural oligoamines with enzymes.
The goal of the present study was to determine the thermodynamic parameters of CTOA complexes and their relation with the activity and stability of the enzyme in waterorganic solvent systems.
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Materials and methods |
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The complexes of -chymotrypsin (CT) (Sigma, EC 3.4.21.1) with spermine, spermidine and putrescine (all from Sigma) were prepared by mixing solutions of the enzyme and OA, both dissolved in 5 mM aqueous MOPS buffer (Sigma), pH 7.45. The enzyme concentration was 0.2 mM. The OA concentration was varied in the range 0.520 mM.
Titration of active sites in preparations of -chymotrypsin
The concentration of active sites in native and modified CT and in enzymeOA complexes was determined spectrophotometrically by using titration with N-trans-cinnamoylimidazole (Sigma) at 25°C and pH 5.05 (Schonbaum et al., 1961; Kudryashova et al., 1997
).
Determination of catalytic activity in binary watercosolvent mixtures
An aliquot of a stock aqueous solution of free CT (0.2 mM) in 1 mM HCl or its complex with OA was added to a watercosolvent binary mixture containing 5 mM MOPS, pH 7.45, to give a final enzyme concentration of 1.0 mM. Ethanol (Reakhim) and N,N-dimethylformamide (DMF) (Sigma) were used as organic cosolvents. In watercosolvent solutions, the pH was adjusted to 7.45 with 1 M KOH [see elsewhere for a discussion of the applicability of this procedure (Ulbrich-Hofmann and Selisko, 1993)]. The enzymatic reaction was started by adding an aliquot of a 40 mM stock solution of N-benzoyl-L-tyrosine p-nitroanilide (BTNA, Sigma) in 1,4-dioxane (VEB Laborchemie Apolda) to obtain a final substrate concentration of 0.041.0 mM. The initial rate of enzymatic hydrolysis was measured spectrophotometrically by following the formation of the product, p-nitroaniline, at 390 nm (Gladilin et al., 1995
; Kudryashova et al., 1997
; Vinogradov et al., 2001
). The value of the molar extinction coefficient of the product was determined at each concentration of organic cosolvent. The values of the maximum reaction rate (Vmax) were determined from the dependences of the reaction rate on the substrate concentration; the rate of spontaneous substrate hydrolysis was always negligible in comparison with the enzymatic rate (Gladilin et al., 1995
; Kudryashova et al., 1997
; Vinogradov et al., 2001
). High concentrations (up to 400 µM) of oligoamines themselves have no effect on the substrate hydrolysis.
Covalent modification of -chymotrypsin
-Chymotrypsin was acylated with pyromellitic anhydride (Aldrich) according to a procedure described elsewhere (Mozhaev et al., 1988b
). A solution of an anhydride (10500 molar excess with respect to the enzyme) in dimethyl sulfoxide (Sigma) was added at 4°C dropwise during 1 min to 40 mM CT (10 ml) in 0.1 M phosphate buffer, pH 8.0, containing 0.05 M N-acetyl-L-tyrosine (Sigma). The pH of the reaction mixture was kept constant by adding dropwise small portions of 1 M KOH. The reaction proceeded for 2 h and then the enzyme was separated from low molecular mass components of the reaction mixture by dialysis. The modified enzyme was stored in a frozen state for future experiments.
Determination of the modification degree
The number of modified amino groups was determined by following the decrease in free (unmodified) amino groups by their spectrophotometric titration with picrylsulfonic acid (Sigma) on a Beckman Model 25 spectrophotometer as described (Mozhaev et al., 1992).
Circular dichroism measurements
Circular dichroism experiments were carried out with a Jobin Yvon Mark V spectrometer at 20°C with quartz cells of pathlength 1 mm in the far-UV region (200260 nm). The protein concentrations were 0.1 mg/ml. Secondary structure percentage predictions were made using CDNN software (http://bioinformatik.biochemtech.uni-halle.de/cdnn).
Fluorescence spectroscopy
Fluorescence spectra were recorded on an SLM-Aminco SPF-500C fluorimeter at 20°C. For all experiments the excitation wavelength was 295 nm. The protein concentrations were 1 µM. The OA concentration was varied in the range 0.5100 µM.
Determination of stoichiometry and dissociation constants of the CTOA complexes using equilibrium dialysis
The complexes of CT with OA were prepared according to the procedure described above. For each OA studied (spermine, spermidine and putrescine) a set of CTOA solutions with different CT/OA ratios was dialyzed against an aqueous solution or 20% v/v ethanol solution in 5 mM MOPS, pH 7.45, for 6 h at 4°C. The CT concentration was 1 µM. The OA concentration was varied from 1 to 400 µM. The concentration of non-bound OA in bulk solution after dialysis was determined using a fluorescence spectroscopic method, which is based on the formation of a fluorescent adduct on the interaction with OPA reagent (containing 1 ml of water, 4 mg of KOH, 50 mg of H3BO3, 3 µl of a 30% aqueous solution of Brij-35, 3 µl of mercaptoethanol, 0.4 mg of o-phthalic dianhydride (Sigma) solution in 5 µl of methanol) according to procedure described by Sparthas et al. (Sparthas et al., 1982). To determine the thermodynamic constants and stoichiometry of the complexes, the analysis of equilibrium dialysis data was performed in Scatchard coordinates.
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Results |
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Catalytic activity of CT in complexes with OA in waterorganic solvent systems
In aqueous solution complex formation with OA does not affect the activity of CT (Figure 1) and also does not lead to any changes in the active site concentration in CT (for all CT preparations studied in this work the content of active enzyme in aqueous medium was about 75%).
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The Michaelis constant of an enzyme, KM, is also known to be dependent on the composition of the medium (Fink, 1974; Maurel, 1978
; Nagamoto et al., 1986
; Mozhaev et al., 1989
). For free CT and all CTOA complexes studied, KM increases with increase in organic solvent concentration (Figure 2
). Such a tendency observed for KM does not seem surprising, since binding of CT substrates in the enzyme active site is mainly guided by hydrophobic interactions (Maurel, 1978
; Mozhaev et al., 1989
). On the other hand, addition of an organic cosolvent increases the hydrophobicity of the solution, which begins to complete effectively for the substrate partitioning with the enzyme active site. This factor could contribute to more rapid growth of the KM value for CTOA in comparison with free CT with increase in ethanol concentration. OA molecules contain charged groups, making the microenvironment of the enzyme in the complex with OA more polar.
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The effects of the complex formation depend strongly on the number of electrostatic contacts between the protein surface and OA molecules. The magnitude of the activation and stabilization effects increases with the length of OA molecules and the number of amino groups in them. Both effects are most pronounced in the case of spermine, which has the longest chain and the greatest number of amino groups per molecule among the three OA studied. Such an OA molecule has a better chance of finding suitably located negatively charged counterparts on the protein surface for electrostatic interactions. Further evidence in support of this conclusion comes from the data in Table I, where the distances between carboxylic groups of aspartic and glutamic acids in the molecule of CT and possible variants of two-point binding for each OA with these groups are shown. As one can see, putrescine (molecular length 6.2 Å) has a possibility of forming only two pairs with carboxylic groups in the molecule of CT. More binding sites can be available for the molecule of spermidine (molecular length 11.05 Å), and spermine (molecular length 16.05 Å) has still more additional possibilities for electrostatic binding with carboxylic groups on the protein surface (Table I
). Further, OA having a long chain may form three-point contacts with the molecule of CT, which can contribute even better to enhanced activation and stabilization effects. Another possibility for increasing the number of electrostatic contacts between CT and OA is to introduce additional negatively charged groups on the protein surface. This can be achieved, for example, by covalent modification with pyromellitic anhydride. Such modification enriches the protein surface with about 30 additional carboxylic groups (Kudryashova et al., 1997
):
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where E is the enzyme molecule.
Enrichment of CT with negatively charged carboxylic groups amplifies both activation and stabilization effects of the complex formation with OA. Modification itself accounts for the elevated resistance of CT to inactivation in waterorganic cosolvent mixtures (Kudryashova et al., 1997), but it is the complex formation that provides the main contribution to enhanced stabilization and activation effects (Figure 3
). The influence of CT modification on the activation effect of the complex formation is the most pronounced in the case of the shorter OA, spermidine and putrescine. For example, complex formation with putrescine leads to additional activation (~1.5-fold) and additional stabilization of the enzyme (up to 20% v/v) compared with the complex of non-modified CT with putrescine (Figures 1
and 3
). At 6080% v/v ethanol pyr-CTput retains much higher catalytic activity (100% of aqueous level) compared with the complex CTput (20% of aqueous level). This is not surprising, because in the case of putrescine the modification of CT significantly increases the number of binding sites on the protein surface (Table I
). At the same time, a longer OA, spermine, has sufficient possibility to form multiple interactions even with non-modified CT. In the case of spermine the influence of modification on the protein stabilization is the most significant at high ethanol concentration, where the maximum protection of protein conformation against inactivation is needed. In the system containing 90% v/v of ethanol pyr-CTsp retains a catalytic activity of about 120% of its aqueous level, whereas the native CTsp is already precipitated at such an ethanol concentration (Figure 1
). It should be mentioned that the observed experimental fact of high catalytic activity of the soluble enzyme in the complex with OA in the systems containing high concentrations of polar organic solvents (ethanol or DMF) might be of paramount importance in many practical areas including peptide synthesis.
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The dependence of catalytic activity of CTOA complexes on OA concentration was investigated in the system containing 20% v/v ethanol. At this ethanol concentration the maximum activation effect of complex formation is observed (Figure 1). The dependence shows a complex profile, which reflects the existence of several types of complexes at different OA concentrations (Figure 4
). Addition of a small amount of OA to the system leads to some decrease in the catalytic activity, which is probably caused by the formation of high-affinity complexes (referred to as CTOA1). Probably, it is a strong multipoint interaction between OA molecules and CT surface on the formation of high-affinity complexes (CTOA1) that leads to the distortion of the enzyme conformation. Such multipoint interaction is much stronger in waterethanol mixture than aqueous solution as a result of the decrease in the polarity of the medium. This could explain the fact that the decrease in catalytic activity is observed in 20% ethanol, but not in aqueous solution. An increase in OA concentration up to an 80100 molar excess of OA over CT results in enhanced enzyme activity. This could reflect the formation of the set of low-affinity complexes (further referred to as CTOA2). Perhaps the low-affinity sites are in fact represented by one-point CTOA binding whereas the high-affinity sites reflect multi-point CTOA binding. With further increase in OA concentration (up to ~200 molar excess of OA) the enzyme activity reaches a plateau, which reflects the saturation of the binding sites on the enzyme surface with OA molecules. The maximum level of the enzyme activity increases with the length of OA (in the order putrescinespermidinespermine) (Figure 4A
). Enrichment of the CT surface with negative charges (upon modification with pyromellytic anhydride) leads to an increase in the maximum level of the enzyme activity and a decrease in the saturation OA concentration to 100 µM (Figure 4B
). Thus, an increase in the number of possible electrostatic contacts between CT and OA molecules by increasing either the chain length of OA molecules or the charge density on the protein surface results in an increase in the activation effect of the complex formation in waterorganic solvent mixtures.
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To study the thermodynamic stability and stoichiometric composition of the complexes of CT with OA of different chain length we applied the method of equilibrium dialysis. Analysis of equilibrium dialysis data in a Scatchard plot allows us to discriminate between different binding sites for OA molecules on the CT surface according to their affinity and to determine the dissociation constants and stoichiometry for each type of complex. An example of a Scatchard plot obtained for the CTOA systems in aqueous solution is shown in Figure 5. The population of high-affinity complexes (CTOA1 with average dissociation constant
) and a set of low-affinity complexes (CTOA2 with average dissociation constant
) were discriminated in the CTOA system, as proposed from the analysis of the enzyme activity profile (Figure 4
).
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In the system containing 20% of ethanol the number of OA molecules strongly bound to the CT surface increases ~2-fold compared with aqueous solution (5, 9 and 14 for putrescine, spermidine and spermine, respectively), which is much higher than the theoretically possible number of two-point contacts of OA with CT (Table I). This result points to the distortion of the protein conformation upon complex formation with OA in 20% ethanol solution, which is probably the reason for the decrease in the enzyme activity at low concentrations of OA. While the dissociation constants
changed only slightly in 20% ethanol, the thermodynamic stability of the CTOA2 complexes increased significantly compared with aqueous solution. The values of
could be estimated as 1.53 µM, depending on the OA chain length. The number of OA molecules bound to low-affinity sites on the CT surface can be estimated as 16, 30 and 30 for putrescine, spermidine and spermine, respectively. From global analysis of the catalytic activity profile (as a function of OA concentration) and the data on the thermodynamic stability and stoichiometry of the complexes, one can suppose that it is the occupation of the low-affinity sites on the CT surface by OA molecules that is responsible for the activation effect of the complex formation in waterorganic solvent mixtures.
Fluorescence spectroscopy
Formation of the complex with OA leads to quenching of CT fluorescence. For example, interaction of CT with spermine decreases the fluorescence intensity of the protein by 2530% in aqueous solution and also in 20% ethanol solution (Figure 6). The interval of OA concentration where the quenching of CT fluorescence is observed corresponds to the range of the formation of high-affinity complexes (compare Figures 4
and 6
), while the activation effect is observed at much higher OA concentrations. This fact also indicates that the activation effect of complex formation is observed only after low-affinity sites on the CT surface are occupied.
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The conformational stability of CT in the complex with spermine in waterethanol mixtures was studied using circular dichroism (CD) spectroscopy in the far-UV region. CD spectra for native CT in aqueous solution and in waterethanol mixtures (Figure 7A) are in good agreement with the spectra reported earlier (Kudryashova et al., 1997
, 2001
). Significant changes of CT structure are observed at an ethanol concentration of 30% v/v: the ß-sheet content is increased at the expense of
-helices and ß-turns, as has been found elsewhere (Vinogradov et al., 2001
). The observed increase in ß-sheet content can be attributed to aggregation of the protein, which has been described previously for CT under the given conditions (Kudryashova et al., 1997
).
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Discussion |
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It can be seen from Figure 1 that the dependences of CT catalytic activity on organic cosolvent content are characterized by a pronounced maximum. Such a shape of the curves is obviously due to the competition of two oppositely directed processes: increase in catalytic activity (mainly due to enhancement of the nucleophilicity of functional groups in the enzyme active site in the presence of organic cosolvent) and decrease in catalytic activity because of enzyme denaturation.
The experimental results suggest that two populations of CTOA complexes (of high and low affinity) are likely to be formed in the system depending on the OA/CT ratio. The formation of high-affinity complexes, which is observed at low OA concentrations, is not responsible for enzyme activation. On the contrary, enzyme incorporated in such a complex reveals a slightly decreased catalytic activity. This may be due to distortion of its native conformation as a result of comparatively strong multi-point electrostatic interaction with OA in waterethanol mixtures.
The activation effect is observed only at high OA/CT molar ratios (~100200), e.g. under conditions when low-affinity sites on the CT surface should be occupied by OA molecules. Perhaps the low-affinity sites are represented by single-point CTOA binding, which should leave the protein conformation and dynamics undistorted. At the same time, it is effectively protected against denaturation in waterorganic mixtures by the buffering native enzyme microenvironment. Hence the low-affinity sites are ultimately responsible for the activation effect.
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Acknowledgments |
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References |
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Received October 17, 2002; revised February 20, 2003; accepted March 7, 2003.