1 A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov str., 119991 Moscow, 3 Department of Chemical Enzymology, M.V. Lomonosov Moscow State University, Vorobievy gory, 117899 Moscow and 4 N.M. Emmanuel Institute of Biochemical Physics, 4 Kosygina str.,117977 Moscow, Russia
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Abstract |
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Keywords: -chymotrypsin/chemical modification/kinetics/stabilization/waterorganic mixtures
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Introduction |
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Biocatalysis in non-aqeous media has been the subject of numerous studies (Oldfield, 1994; Tuena de Gomez-Puyou and Gomez-Puyou, 1998
; Halling, 2000
; Klibanov, 2001
). Some enzyme suspensions in non-polar water-immiscible organic solvents were shown be to very stable (Zaks and Klibanov, 1985
). It has been suggested that in more polar solvents, water molecules, essential for stabilizing the native conformation, are stripped from the protein surface (Zaks and Klibanov, 1984
). Several approaches have been proposed to make water molecules bind more tightly or to immobilize the protein in hydrophilic matrices (Khmelnitsky and Rich, 1999
).
Enzymes dissolved in polar organic solvents or in homogeneous waterorganic media usually lose their native conformation and catalytic activity. It is generally believed that the introduction of additional hydrophilic groups on the protein surface may improve its stability in such systems owing to the enhanced ability of the hydrophilized surface to keep the hydration shell and form additional electrostatic interactions, hydrogen bonds or salt bridges (Khmelnitsky et al., 1991; Mozhaev et al., 1996
). On the other hand, hydrophobic groups, if introduced near the hydrophobic region on protein surface, may also contribute to protein stabilization (Lee and Richards, 1971
; Chotia, 1984
). Arnold and co-workers suggested that the replacement of disordered, uncompensated surface charge with hydrophobic residues using site-directed mutagenesis may dramatically improve enzyme stability (Arnold, 1988
; Martinez and Arnold, 1991
). Crambin, a small hydrophobic protein, is an amazing demonstration of natural protein design for non-aqueous media: it is soluble and retains its native structure at very high concentrations of polar organic solvents (De Marco, 1981). Crambin is not soluble in aqueous solutions, but has water-soluble homologs with similarly folded structure (Teeter et al., 1981
). In comparison with these homologs, crambin possesses amino acid substitutions, removing hydrophilic side chains: lysines, arginines and asparagines are almost excluded in crambin.
In recent work, we have performed hydrophobization/ hydrophilization via chemical modification of -chymotrypsin surface amino groups. The aim was to understand how both types of modification affect enzyme activity, structure and stability.
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Materials and methods |
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Acetylation of -chymotrypsin (CT) (Sigma, EC 3.4.21.1) was carried out with pyromellitic anhydride (Aldrich) and succinic anhydride (Reanal) as described previously (Mozhaev et al., 1988
).
Reductive alkylation with glyceric, pentylic, isobutyric and acetic aldehydes (Reanal) was carried out according to Mozhaev et al. (Mozhaev et al., 1992). Modification with a mixture of glyceric and pentyl aldehydes was made in exactly the same way (a 100 molar excess of each aldehyde with respect to the enzyme was taken).
Determination of the degree of modification
The degree of CT modification was calculated from the number of amino groups in it which reacted with trinitrobenzenesulfonic acid (TNBS) as compared with the unmodified enzyme (Fields, 1971).
Titration of -chymotrypsin active sites
The concentration of active sites in all CT samples was determined with N-trans-cinnamoylimidazole (Sigma) as titration reagent (Schonbaum et al., 1961).
Other procedures
Inhibition with phenylmethanesulfonyl fluoride (PMSF) was performed as described (Fahrney and Gold, 1963).
The enzymatic activity of -chymotrypsin in binary waterorganic mixtures was determined with N-benzoyl-L-tyrosine-p-nitroanilide (BTNA) (Sigma) and with 4-methylumbelliferyl-p-trimethylammonium cinnamate chloride (MUTMAC) as described previously (Kudryashova et al., 1997
).
Calculations of hydrophobicity of modifiers and of change of hydrophobicity of modified CT were made according to Mozhaev et al. (Mozhaev et al., 1992).
Differential scanning calorimetry (DSC) measurements were made essentially as described elsewhere (Grinberg et al., 2000). Protein solutions for calorimetric measurements were dialyzed against corresponding media for at least 5 h at 4°C. Buffer solution was 20 mM MOPS, pH 7.45. The protein concentration after dialysis was determined spectrophotometrically using the extinction coefficient E280 = 50 000 M1 cm1 (Volini and Tobias, 1969
). The concentration of protein solutions used for calorimetric measurements was 0.51.2 mg/ml. DSC measurements were carried out with a DASM-4 adiabatic differential scanning microcalorimeter (Biopribor, Puschino, Russia) at a heating rate of 1°C/per min and at extra pressure 1 atm. Primary data processing was performed using NAIRTA software (Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia).
A quantitative thermodynamic analysis should be based on equilibrium studies. Nevertheless, in some cases one can use this analysis for practically irreversible processes (Privalov and Potekhin, 1986). The thermal denaturation of CT is known to be irreversible. To minimize the interference of irreversible denaturation, all measurements were made under the same conditions. We aimed to compare the calorimetric parameters of modified and native samples of CT.
Circular dichroism experiments were carried out with a Jobin Yvon Mark V spectrometer at 25°C with quartz cells of pathlength 1 mm in the far-UV region (200260 nm). The protein concentrations were 0.10.2 mg/ml. The secondary structure percentage predictions were made using CDNN software (http://bioinformatik.biochemtech.uni-halle.de/cdnn).
Polyacrylamide gel electrophoresis was performed under denaturing conditions according to Laemmli (Laemmli, 1970).
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Results |
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We found that both surface hydrophilization and hydrophobization enhance protein stability against denaturation in waterorganic mixtures. This effect was expressed in (i) broadening of the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value and (ii) the increase in the organic cosolvent concentration range at which the residual enzyme activity is observed.
In waterorganic solvent mixtures native (non-modified) CT is relatively unstable (Mozhaev et al., 1989; Kudryashova et al., 1994
; Gladilin et al., 1995
). It is almost inactive at 50% (v/v) ethanol (EtOH), which is in agreement with data from other groups (Sato et al., 2000
); in 40% (v/v) dimethylformamide (DMF) CT is completely inactive owing to denaturation. At moderate organic cosolvent concentrations (up to 20%, v/v) native CT is more active than in a water environment (Figures 1 and 2
). Further, the catalytic activity is gradually decreased to complete inactivation.
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Normally, not more than 20% of CT active sites were lost in the course of modification. The catalytic properties of modified samples were not dramatically different from native enzyme owing to several experimental facts. (1) The hydrolysis of two substrates differing in the rate-limiting step, BTNA and MUTMAC, was investigated. The rate-limiting step for BTNA is acyl-enzyme formation (West et al., 1990) and for MUTMAC it is deacylation (Maurel, 1978
). The catalytic activity of modified samples in aqueous solution does not change from native CT by more than 50%, taking into account the percentage of the active sites. In waterorganic media the modified samples demonstrate similar trends to the native enzyme, but possess higher stability (Figures 1 and 2
). (2) The pH dependence of catalytic activity for modified chymotrypsins is similar to that for native chymotrypsins (data not shown). (3) All modified samples were characterized using SDSPAGE and no difference from the native form was observed (data not shown). This also indicates that intermolecular `sewing', which could be expected in the case of pyromellitic anhydride, does not take place. (4) The Km values of modified samples are the same order of magnitude as that for the native enzyme. The Km values of modified and native CT samples behave similarly: they increase with organic cosolvent concentration (data not shown).
We found that the values of the stabilization effect, expressed by organic cosolvent concentration at which enzyme activity returns to its initial value (C100), can be correlated with the increment of hydrophobicity (G) introduced by the modification (Figure 3
). The values of (
G) were calculated from Hansch hydrophobic increments of functional groups and atoms (Mozhaev et al., 1992
; Leo et al., 1971
) and are given in Table I
. In the case of hydrophilic and hydrophobic modifications at small and moderate increments (
G), the stabilization effect is proportional to the amounts of hydrophobicity introduced. It is interesting that left and right halves of the curve, corresponding to hydrophobization and hydrophilization of enzyme surface, are almost symmetric about the y-axis (Figure 3
). At higher
G the stabilization effect is almost independent of further increases in the increment.
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Circular dichroism
The samples analyzed by far-UV CD spectroscopy, glyc13CT (hydrophilic modification) and ibut7CT (hydrophobic modification), are fairly similar considering the catalytic activity in waterethanol media (Figure 2). They are much more stable against inactivation than the native form. However, from the CD spectra we found that the structural properties of the analyzed samples in aqueous solution and in waterethanol media are very different.
The CD spectrum for native CT (not shown) was similar to the published spectrum (Gorbunoff, 1971). For comparison, the values according to theoretical data are also presented. Upon addition of ethanol up to 20% (v/v) practically no changes in the CD spectra are observed. The values of secondary structure contents vary within the estimation error (Table III
). The addition of more ethanol (30%, v/v) to the media gives rise to considerable changes in the structure: the ß-sheet content is increased mainly at the expense of
-helix and ß-turn. Very similar changes take place at higher ethanol concentrations (50 and 70%, v/v). The observed increase in ß-sheet content can be attributed to aggregation of the protein, which is known for CT under the given conditions (Kudryashova et al., 1997
). It is worth noting that the decrease in catalytic activity is the sharpest between 25 and 30% (v/v) ethanol (Figure 2
). Hence the inactivation of CT in waterethanol mixtures can be accounted for by aggregation. The formation of ß-structures and loss of activity in 50% ethanol were reported for native CT also by another group (Sato et al., 2000
).
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For DSC studies we selected glyceric aldehyde-modified CT with degree of modification 13 (hydrophilic modification, glyc13CT); and pentylic aldehyde-modified CT with degree of modification 11 (hydrophobic modification, pent11CT). As we mentioned, these samples were considerably more stable than native CT in respect of retention of catalytic activity in waterDMF mixtures (Figure 1). The parameters obtained by DSC indicate that in aqueous media both modifications lead to the formation of CT structure with higher conformational stability (Table II
).
We also followed the behavior of pent11CT and glyc13CT in waterDMF media using DSC techniques. For both modified and native CT, the addition of DMF causes an almost linear decrease in denaturation temperature and a non-linear decrease in enthalpy of denaturation. For native CT the excess heat capacity function contains a single peak, which apparently corresponds to a single cooperative system (Privalov, 1979). However, for both modified samples, particularly at 1020% (v/v) DMF, the experimental excess heat capacity function shows the presence of several fractions or quasi-independent structural components (Privalov, 1981
). We deconvoluted the experimental excess heat capacity functions as a superposition of several independent components and calculated the parameters for each of them (Figure 7
).
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Discussion |
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Replacements of surface-charged amino acids with hydrophobic ones using site-directed mutagenesis was reported to improve enzyme stability substantially (Arnold, 1988; Martinez and Arnold, 1991
; Martinez et al., 1992
; van den Burg et al., 1994
). This approach works if the replaced hydrophilic residue is not crucial for maintaining salt bridges and a hydrogen-bond network. But how could such explanation be extended to the case of chemical modifications, where the sites of modification are not known?
From the chemical point of view, the reactivity of amino functions involved in multiple hydrophilic interactions will be lower. Hence they would be less susceptible to chemical modification. If that is so, uncompensated, unsatisfied surface residues (lysines and N-terminal amino acids) would have the highest reactivity. If our assumption is correct, all these residues are the first candidates for chemical modification. Substituted with a hydrophobic substance, they can be expected to contribute to protein stabilization in waterorganic mixtures. However, if this mechanism was really working in our case, one could expect the stabilizing effect not only for pentylic and isobutyric aldehydes, but also for acetic aldehyde. Our data indicates that this is not so no effect of the latter was observed. Therefore, to make the protein more stable it is not sufficient just to replace the unfavorable surface charge.
In our case, it could be supposed that additional hydrophobic groups introduced during modification create additional interactions with unfavorable hydrophobic regions on the protein surface. Apparently, this must contribute to protein stabilization (Lee and Richards, 1971; Chotia, 1984
). The hydrophobic increment of acetic aldehyde is perhaps not sufficient to build such interactions.
Another possibility is that in the course of modification the distribution of modificator molecules around the protein globule is not even: more hydrophobic molecules would preferentially react with amino groups adjacent to uncompensated hydrophobic regions on the protein surface. If so, we have a kind of self-adjusting system, where the modifier `finds' the way to attach near the uncompensated region. Similar suggestions can be put forward for hydrophilic modification. For the examination of our hypothesis we performed the modification of CT with a mixture of glyceric and pentylic aldehydes (glycpentCT) taken in equal amounts. As mentioned in the Results section, the total degree of modification of this sample was determined as 11; also considering the reactivity with glyceric and pentylic aldehydes, the degrees of modification for them should be equal. Hence the G value should be about 40 kJ/mol (see Table II
). It is amazing that despite such a small increment, glycpentCT appeared to be the most stable of all modified samples studied in recent work. This is an indication that the most efficient stabilization is not achieved by hydrophilization or hydrophobization itself, but by `successful' modification. Perhaps in glycpentCT the structure has been optimally readjusted, so that both stabilization mechanisms are working.
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Notes |
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Acknowledgments |
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References |
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Received February 16, 2001; revised June 8, 2001; accepted June 18, 2001.