Abstract: Kinetic and structural studies have been made on the effect of glyoxime (GO) and dichloroglyoxime (DCGO) on the activity and the structure of lysozyme in 100 mM potassium phosphate buffer, pH 7.0, using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy techniques. GO and DCGO act as an uncompetitive inhibitors with Ki = 99 and 52 µM, respectively. Circular dichroism studies show that the secondary structure of the enzyme in the presence of different concentrations of GO and DCGO does not show considerable change. Kinetic results show that at low concentration of GO (0.12-120 µM) and DCGO (0.07-64 µM) considerable inhibition of enzyme could be seen, but the fluorescence data show that there is not noticeable change in the tertiary structure of lysozyme at low concentration of inhibitors. Also, these results indicate considerable decrease in the tertiary fold of the lysozyme at high concentrations of GO and DCGO espetially dichloroglyoxime. Results show that, lysozyme in the presence of high concentration of GO (1200 µM) and DCGO (640 µM), presents structural characteristics of a molten globule like state.
Introduction
α-dioxime derivations have many applications in the industry and agriculture, for example, for chemical sensors that are used for special elements and for pesticides, fungicides and bactericides. Remarkable activity against gram-positive and gram-negative bacteria as well as certain yeast was observed for dichloroglyoxime. In addition, these substances effect biological systems
adversely. For example, dimethyl glyoxime causes irritation of the eye, skin, digestive and respiratory systems and moreover, proven dangerous if ingested. Experimental results show that these substances are mutagens (Khalili et al., 1986; Bing et al., 1999; Ayres et al., 2002). Lysozyme (EC 3.2.1.17) is found in various animal and plant tissues, e.g., in tear liquid and chicken egg white. It functions as an antibacterial agent catalyzing the hydrolysis of a major cell-wall polysaccharide of certain bacteria (Ovchinikov, 1996).
This enzyme hydrolyses β(1→4) glycosidic linkage from NAM (N–acetylmuramic acid) to NAG (N-acetylglucosamine) in the alternating NAM-NAG polysaccharide component of cell wall peptidoglycan of bacteria. Lysozyme is single domain protein and the active site is located just in the cleft (Ovchinikov, 1996; Schomburg et al., 1991; Smith et al., 1993). Hen egg white lysozyme is one of the most studied and best characterized globular proteins. It was the first enzyme to have its structure determined by X-ray diffraction and has ever since been extensively used as a system in which the underlying principles of protein structure, function, dynamics and folding can be studied through both experimental theoretical approaches (Smith et al., 1993; Matagne et al., 1998; Alizadeh et al., 2003; Godjaev et al., 1998; Fujita et al., 1995; Iwase et al., 1999; Johannesson et al., 1997; Schwalbe et al., 2001; Kristiansen et al., 1998).
Lysozyme binds to various small ligands which mimic the interaction of the acetamido group of its natural substrates within the active cleft of the enzyme (Johannesson et al., 1997; Imoto et al., 1972). Glyoxime (GO) and dichloroglyoxime (DCGO) were synthesized (Scheme 1) in our laboratory (Kakanejadifard et al., 2003, 2004) and in the present research we describe the catalytic behavior and structural changes of lysozyme in the presence of GO and DCGO.
Materials and Methods
Materials
The hen egg white lysozyme and Micrococcus lysodeikticus were purchased
from Sigma (St. Louis, Mo, USA). The enzyme was homogenous on SDS-PAGE. All
other chemical reagents were from Merck (Darmstadt, Germany) and reagent grade.
The solutions were prepared in double distilled water. Glyoxime was obtained
by the condensation of glyoxal and hydroxylamine in water at 0°C and dichloroglyoxime
were synthesized by chlorination of glyoxime in ethanol in our laboratory (Kakanejadifard
et al., 2003, 2004; Coburn, 1968; Willer et al., 1985; Kanno
et al., 1995).
Determination of Enzymatic Activity and Protein Concentration
Enzymatic activity was determined using Micrococcus lysodeikticus
as substrate in 100 mM potassium phosphate buffer, pH 7.0. One enzymatic unit
is equal to a decrease in turbidity of 0.001 per minute at 450 nm at pH 7.0
under specified conditions (Worthington, 1993). The protein concentration was
determined by Lowry et al. (1951) method.
Circular Dichroism (CD) Measurements
CD spectra were recorded on a JASCO J-715 spectropolarimeter (Japan) using
solutions with protein concentrations varying from 0.15 (far-UV) to 2 mg mL-1
(near-UV). The results were expressed as molar ellipticity, [θ] (deg cm2
dmol-1), based on a mean amino acid residue weight (MRW) assuming
its average weight for lysozyme to be equal to 111.5. The molar ellipticity
was determined as [θ] = (θx100MRW)/(cl), where c is the protein concentration
in milligrams per milliliter, l is the light path length in centimeters and
θ is the measured ellipticity in degrees at a wavelength λ. The instrument
was calibrated with (+)-10-camphorsulfonic acid, assuming [θ] 291
= 7820 deg cm2 dmol-1 (Schippers et al., 1981)
and with JASCO standard nonhydroscopic ammonium (+)-10-camphorsulfonate, assuming
[θ] 290.5 = 7910 deg cm2 dmol-1 (Takakuwa
et al., 1985). Noise in the data was smoothed using the JASCO J-715 software,
including the fast Fourier-transform noise reduction routine which allows enhancement
of most noisy spectra without distorting their peak shapes (Protasevich et
al., 1997; Ataie et al., 2004; Khajeh et al., 2001).
Intrinsic Fluorescence Measurements
The fluorescence emission spectra of the enzyme were performed in a Perkin-Elmer
luminescence spectrometer LS50B. The spectra were measured in 100 mM phosphate
buffer, pH 7.0 and the final concentration of 10 μM for lysozyme. The fluorescence
emission was scanned between 300 and 400 nm with an excitation wavelength of
280 nm.
Aggregation Measurements
Lysozyme at a concentration of 0.5 mg mL-1 in 100 mM phosphate
buffer, pH 7.0, in the presence of different concentrations of GO and DCGO were
placed in Perkin-Elmer luminescence spectrometer LS50B cuvette. The excitation
and emission monochromators were set at 350 and 355 nm with the band passes
of 1.5 nm and the extent of light scattering was monitored. The scattering effect,
due to different concentrations of GO and DCGO, was corrected in all relevant
cases.
Results presented in this study are the mean from at least three repeated experiments in a typical run to confirm reproducibility.
Results and Discussion
Catalytic Parameters of Lysozyme in the Presence of α-dioxime Derivatives
The activity of lysozyme has been estimated in the presence of different
concentrations of glyoxime and dichloroglyoxime. The enzyme activity decreases
as the concentration of GO and DCGO increase (Fig. 1).
| Fig. 1: | Dose-response curves of lysozyme activity as a function of different concentrations of glyoxime (a) and dichloroglyoxime (b) |
| Table 1: | Kinetic parameters for lysozyme in the absence and presence of glyoxime and dichloroglyoxime |
| Fig. 2: | Lineweaver-Burk plots of lysozyme in the absence and presence of different concentrations of glyoxime (a) (1) 0, (2) 0.12, (3) 120 μM and dichloroglyoxime (b) (1) 0, (2) 6.4 and (3) 64 μM |
| Fig. 3: | Plot of 1/K’m versus different concentration of inhibitors; K’m is the apparent Michaelis constant in the presence of inhibitor glyoxime (a) and dichloroglyoxime (b) |
Effects of the inhibitors on lysozyme is time-independent and after effects of inhibitor on lysozyme is eliminated with dialysis against buffer, activity of the enzyme is recovered (data not shown). These results show that lysozyme inhibition with GO and DCGO is reversible. To compare the inhibition effects of GO and DCGO, IC50 values of these compounds based on Fig. 1 were evaluated. These values are 60 and 95 μM for DCGO and GO, respectively. Our results indicate that inhibition strength of DCGO is higher compared to GO. This could be due to the presence of chloride in DCGO and its high electronegativity. Figure 2 shows lineweaver-Burk plots of lysozyme in the absence and the presence of different concentrations of GO and DCGO.
| Fig. 4: | Far-UV CD spectra of lysozyme in the absence and presence of the different concentrations of glyoxime (a) and dichloroglyoxime (b). (1) in the absence and others in the presence of glyoxime and dichloroglyoxime |
Vmax and Km of the enzyme, in the presence and absence of inhibitors, were obtained and the type of inhibition was discussed. Vmax and Km decreased as the concentration of these substances increased (Table 1). These results indicate that inhibition mechanism is uncompetitive (Copeland, 2000). The secondary plot was prepared to calculate Ki (Fig. 3) (Copeland, 2000; Saboury et al., 2002) and subsequently, the inhibition constants for GO and DCGO were shown to be 99 and 52 μM, respectively. These results confirm the finding that inhibition strength of DCGO is higher than GO.
Comprehensive structural studies have been reported for GlcNAc oligosaccharides, urea and DMSO all of which bind to the active site of the enzyme both in crystal (Blake et al., 1994) and in solution (Cohen and Jardetzky, 1968; Lumb and Dobson, 1992). DMSO, at low concentration, binds to the active cleft and the protein surface and at high concentration, disrupts the tertiary fold of lysozyme (Johannesson et al., 1997). GO and DCGO can form as many hydrogen bonds with the enzyme as N-acetyl glucose amine or urea, the similarity of their structures with acetamido group suggests that they can bind to the active cleft and the enzyme surface. Our results show that GO and DCGO is able bind to enzyme-substrate [ES] complex.
Circular Dichroism and Fluorescence Measurements of GO- and DCGO- Induced
Equilibrium Denaturation
The far-UV-CD spectra of the lysozyme obtained in phosphate buffer, pH 7.0
at different GO and DCGO concentrations are shown in Fig. 4.
Percentage of secondary structures in lysozyme, in the absence and presence
of glyoxime and dichloroglyoxime, are displayed in Table 2.
Upon the addition of the mentioned inhibitors (0.00064-640 μM of DCGO and 0.0012-1200 μM of GO) CD spectra of lysozyme show a very slight increase in the negative ellipticity at 208 and 222 nm with respect to that of the proteins in the buffer only. In fact, these results show that the enzyme ellipticity at 222 nm, characteristic of the α-helical conformation, does not change in the presence of GO and DCGO, indicating that a native-like secondary structure persists even at 640 μM of DCGO and 1200 μM of GO. The fluorescence emission spectra of lysozyme in phosphate buffer, pH 7.0 at different concentrations of GO and DCGO are shown in Fig. 5. The observed effect of initial increase of lysozyme fluorescence could be interpreted as the consequence of inhibitor binding in the cleft region, in the proximity of Trp62, Trp63 or Trp108, which decreases the accessibility of indole rings for water molecules. Upon addition of these substances, the tryptophan fluorescence of the lysozyme gradually decreases.
| Table 2: | Secondary structure percentage of lysozyme in the absence and presence of glyoxime and dichloroglyoxime |
| Fig. 5: | Fluorescence emission spectra of lysozyme in the absence and presence of different concentrations of glyoxime (a) (1) 0, (2) 1.2x10-3, (3) 1.2x10¯1, (4) 12, (5) 580, (6) 870 and (7) 1200 μM and dichloroglyoxime (b) (1) 0, (2) 6.4x10-4, (3) 6.4x10¯2, (4) 6.4, (5) 320, (6) 480 and (7) 640 μM |
The decrease is observed in the fluorescence intensity, suggesting that at high concentrations of GO (580-1200 μM) and DCGO (320-640 μM), there is a change in the tertiary structure of the protein, resulting in the exposure of the buried tryptophans to the polar solvent. In other words, the intrinsic fluorescence reduction along with the red shift shows that the enzyme has denatured at high concentrations of glyoxime and dichloroglyoxime. The tertiary fold reduction of lysozyme at high concentrations of these reagents (especially DCGO) could probably be the consequence of water structure breaking down by the formation of strong hydrogen bonds between these reagents and the water molecules. The expansion of the protein structure, occurs at the 1200 and 640 μM of GO and DCGO, respectively. Previously, the neutron diffraction and the computer simulation studies of DMSO-water mixtures revealed similar results (Soper et al., 1992; Luzar et al., 1993; Iwase et al., 1999). Present results indicate that at high concentrations of both reagents, lysozyme adopts the features of the molten globule state, with substantial secondary structure and at the same time, the tertiary structure is less organized than that of the native state (Hosseinkhani et al., 2004; Asghari et al., 2004; Boren et al., 1999).
Aggregation Measurements
Light scattering methods provide a sensitive measure of aggregate formation
in protein solutions (Rajaraman et al., 1996). Figure 6
shows the light scattering profile of the lysozyme (0.2 mg mL-1)
as a function of different concentrations of GO and DCGO.
| Fig. 6: | Light scattering profiles of lysozyme as a function of different concentrations of glyoxime (a) and dichloroglyoxime (b) |
Results indicate that in the presence of DCGO, significant aggregation takes place since the folding or unfolding intermediates of proteins, including the molten globule intermediates, expose hydrophobic surfaces and create a tendency to aggregate (Fig. 6).
Thus, lysozyme activity, at low concentrations of GO and DCGO is inhibited by a mechanism that is uncompetitive and in the presence of high concentrations of these reagents, the enzyme displays structural characteristics of a molten globule like state. Several proteins in this state have been shown to be sticky and prone to aggregation (Goto, 1991; Sivaraman et al., 1997). However, in the light of the aggregation data obtained, the probability of molten globule state induction for lysozyme is higher in the presence of DCGO.
Acknowledgment
The financial support by Tarbiat Modares University is gratefully acknowledged.