| BackgroundSuperoxide dismutases catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. These enzymes play an important antioxidant defense role in various organisms exposed to oxygen. Five SOD types that have different metallic ion species present in their active site have been identified. Copper/zinc-containing intracellular SODs (Cu/Zn-SOD) are found in the cytosol and also in the chloroplasts of plants and algae. Copper/zinccontaining extracellular SODs are secreted by many types of cells and are anchored to the plasma membrane or circulate in the extracellular fluids. Manganese-containing SODs (Mn-SODs) are synthesized in the cytosol and imported post-translationally into the mitochondrial matrix.Iron-containing SODs (Fe-SODs) are found in eukaryotes and are homologous to iron-dependent bacterial SODs, and are also present within the chloroplasts of some plants. Lastly, nickel-containing SODs (Ni-SODs) have a hexameric structure and are built from right-handed 4-helix bundles, each containing N-terminal hooks that chelate a nickel ion. These five classes of SOD enzymes catalyze the same reaction. Furthermore, the two forms of Cu/Zn-SOD (intracellular and However, Cu/Zn-extracellular) share high sequence and structural homology. SOD is structurally distinct from Cu/Zn-SOD and Fe-SOD. Cu/Zn- SOD and Fe-SOD share a high degree of amino acid sequence and structural homology. They are highly homologous to one another at the primary, secondary, and tertiary levels and have virtually identical metal binding and catalytic sites. Despite this extensive homology, Cu/Zn-SOD and Fe-SOD are only active with their cognate metal ions. Substitution of iron into Cu/Zn-SOD or viceversa alters the redox potential of the enzyme’s active site and prohibits superoxide disproportionation. Due to the various types of SODs and their important functions, SODs have been used as biomarkers of cellular stress and toxicity in different aquatic organisms such as mollusks, cnidarians, and fish. SODs are also being increasingly used as model proteins to study protein folding and unfolding.Tyrosinase is a critical enzyme that has multi-catalytic functions in the melanosynthetic pathway, and it is distributed ubiquitously in organisms.Two Cu2+ ions individually connected with three histidines at the active site are charged in their cupric or cuprous state and are directly involved in different catalytic reactions via the oxy-, deoxy-, and met-states. The tyrosinase catalysis mechanism is very complex as this enzyme can catalyze multiple reactions, and the mechanism therefore needs to be investigated from different sources using various kinetic methods. Because the crystallographic structure of tyrosinase has not yet been clearly solved, there is little data available to investigate structure-function relationships in this enzyme. Tyrosinase deficiencies are directly associated with pigmentation disorders in mammals and cause a browning effect in vegetables. Tyrosinase also participates in cuticle formation in insects. Therefore, regulation of the enzymatic activity of tyrosinase has been the focus of investigation due to its potential applications in medicine, cosmetics, and agriculture.There are very few reports of regulation of the enzymatic activity of tyrosinase via conformational changes. The major strategy for tyrosinase inhibition has been chelating of the copper ions at the active site.MethodTo measure Cu/Zn-SOD activity, we used a spectrophotometric assay. Activity was calculated based on the autoxidation of pyrogallol, which can be monitored by measuring absorbance at 325 nm every minute. Reactions were performed in a typical reaction volume of 1 ml to which 10μl of enzyme solution was added. The activity of the enzyme was assessed by measuring absorbance using a Perkin Elmer Lambda Bio U/V spectrophotometer.Cu/Zn-SOD was denatured by incubation in 50 mM Tris-HCl (pH 8.2) containing various concentrations of TFE for 3 h at 25℃. The TFE concentration was varied from 0 to 60%(v/v). Fluorescence emission spectra were measured using a Jasco FP750 spectrofluorometer with a 1-cm path-length cuvette. An excitation wavelength of 280 nm was used for the tryptophan fluorescence measurements, and the emission wavelength ranged between 300 and 410 nm. The changes in ANS-binding fluorescence intensity of Cu/Zn-SOD were studied by labeling the enzyme with 40 μM ANS for 30 min prior to measurement. An excitation wavelength of 380 nm was used for the ANS-binding fluorescence, and the emission wavelength ranged from 400 to 650 nm. Circular dichroism (CD) spectra were recorded on a Jasco 725 Spectropolarimeter. The sample cell path length was 22 mm. CD measurements were carried out according to the manufacturer’s instructions. The change in α-helical content of Cu/Zn-SOD was measured as described previously. Among the many tools available for in silico proteinligand docking, we used DOCK6.3 because of its automated docking capability. This program performs ligand docking using a set of predefined 3D grids of the target protein and a systematic search technique. The 3D structure of Cu/Zn-SOD was constructed from the PDB entry,2SOD. The original structure of TFE was derived from the Pub-Chem database (Compound ID:6409, http://www.pubchem.org). Prior to the docking procedure, we performed the following steps:1) conversion of 2D structures to 3D structures,2) calculation of charges,3) addition of hydrogen atoms, and 4) location of pockets.The assay for tyrosinase was performed spectrophotometrically. Reactions were performed in a typical reaction volume of 1 ml to which 10μl of enzyme solution was added to measure tyrosinase activity. The activity and absorption were measured with a Perkin Elmer Lambda Bio U/V spectrophotometer. The v value used in the present study indicates the change in absorbance at 492 nm per minute. Circular dichroism (CD) spectra were recorded on a Jasco 725 spectropolarimeter. The sample cell path length was 22 mm. Fluorescence emission spectra were measured with a Jasco FP750 spectrofluorometer using a 1-cm-path-length cuvette. An excitation wavelength of 280 nm was used for the tryptophan fluorescence measurements, and the emission wavelength ranged between 300 and 410 nm. Changes in the extrinsic fluorescence intensity were studied by labeling with 40μM ANS for 30 min prior to measurement.An excitation wavelength of 390 nm was used for the ANS-binding fluorescence, and the emission wavelength ranged from 400 to 520 nm. The MODELLER program automatically provides an all-atom model using alignments between the query sequence and known homologous structures. We retrieved the known homologous structures of tyrosinase from the Protein Data Bank (PDB) (http://www.pdb.org/) and found that four entries (PDB entry=lwxc, lxom,2oic,2oid) were suitable structural templates (average 26% sequence identity) and were partial tyrosinase homologs. A sequence alignment between tyrosinase and the templates was constructed by ALIGN2D in the MODELLER package. Based on the sequence alignment, the 3D structure of tyrosinase was constructed with a high level of confidence. We subsequently calculated the conformational energy of the structural model of tyrosinase using the discrete optimized protein energy (DOPE) score as a stability metric. Among the many tools available for in silico protein-ligand docking, AutoDock4 and DOCK6 are the ones most commonly used because of their automated docking capability. The programs both perform ligand docking using a set of predefined 3D grids of the target protein; however, AutoDock uses a random search technique while DOCK uses a systemic search technique. Therefore, we used two slightly different approaches to evaluate the docking of tyrosinase and TFE. The original structure of TFE was derived from the PubChem database (compound ID 6409) (http://www.pubchem.org/). To prepare for the docking procedure, the following steps were taken:(1) conversion of 2D structures to 3D structures, (2) calculation of charges, (3) addition of hydrogen atoms, and (4) location of pockets. For these steps, we used the fconverter program of the J-Chem package (http://www.chemaxon.com/) and OpenBabel (http://openbabel.sourceforge.org/).RESULTSWe assayed the kinetics of Cu/Zn-SOD at equilibrium and during TFE-induced unfolding. Cu/Zn-SOD activity was sustained at concentrations of TFE lower than 20% and was gradually inactivated by higher TFE concentrations in a dose-dependent manner, although there was still some residual activity. Even 60% TFE inhibited Cu/Zn-SOD activity by less than 40%. These results indicate that Cu/Zn-SOD activity is relatively resistant to disruption by TFE. To evaluate the inactivation kinetics and rate constants, timeinterval measurements were performed. The time-course of Cu/Zn-SOD inactivation in the presence of 60% TFE was recorded. Enzyme activity decreased gradually over time. The microscopic inactivation rate constants (ka) were calculated from the semilogarithmic plot with the reaction course plotted as monophasic. The rate constant for 60% TFE was 0.343±0.10×10-3s-1 (n= 2). This result suggests that Cu/Zn-SOD inactivation by TFE follows a first-order kinetic process regardless of the fact that the inactivation degree was low.To compare changes in activity with structural changes, we measured tertiary structural changes in Cu/Zn-SOD in the presence of TFE using intrinsic and ANS-binding fluorescence measurements. The intrinsic fluorescence changes monitored by the red-shift in the peak wavelength in the spectra as well as intensity changes revealed that TFE induced significant Cu/Zn-SOD unfolding; as the concentration of TFE increased from 0 to 60%, the fluorescence peak changed from 330.5 to 336.5 nm. A plot of peak wavelength versus [TFE] revealed a sigmoidal relationship. The kinetics of Cu/Zn-SOD unfolding by TFE were also monitored. Semilogarithmic plots showed that the unfolding process also followed first-order kinetics with a rate constant (ku) of 0.71×0.07×10-3s-1 (n= 2). These results indicated that unfolding of Cu/Zn- SOD is synchronized with inactivation of the enzyme. Interestingly, Cu/Zn-SOD activity remained largely intact at lower than 20% TFE, and activity was stable at this TFE concentration. However, overall structural changes in Cu/Zn-SOD occurred at this concentration of TFE, indicating that the TFE binding sites are not located in the activesite pocket where copper- and zinc-mediated catalysis occurs. Furthermore, these results imply that the active site of Cu/Zn-SOD is not as flexible as its overall tertiary structure. Next, we monitored the changes of hydrophobic surface of Cu/Zn-SOD in the presence of TFE. ANS-fluorescence intensities changed in a complicated manner in response to a range of TFE concentrations (5 to 60%). Native Cu/Zn-SOD had a relatively high hydrophobic surface content that reacted directly with the ANS dye; however, the hydrophobic surfaces of Cu/Zn-SOD did not become more significantly exposed during TFE-mediated unfolding. In general, ANS dye can bind to hydrophobic amino acid residues, and is therefore used to monitor disruption of the tertiary structure of a protein in the presence of an inactivator. We found that TFE at concentrations lower than 20% significantly decreased the hydrophobic surface of Cu/Zn-SOD, but concentrations of TFE up to 60% increased the surface hydrophobic content to a level similar to that seen in the native enzyme.As the concentration of TFE increased, the overall α-helical content changed in a non-dose-dependent manner. Although TFE is known to stabilize helices within proteins, it actually decreased the extent of secondary structure in Cu/Zn-SOD. Specifically, the measurements at 222 nm indicated that the overall helical content decreased with increasing TFE concentration, but in a complex manner. When the TFE concentration was increased to 20%, the helical content decreased drastically compared to the native state; however, when the TFE concentration was increased to 30%, the helical content recovered to 60% of the native state helical content and then gradually decreased with increasing TFE concentrations up to 60%. The changes in secondary helical structure appear to be associated with changes in the hydrophobicity of Cu/Zn-SOD; a concentration of 20% TFE was the point at which noticeable changes occurred in both the hydrophobicity and secondary structure experiments.Because a crystallographic structure of Cu/Zn-SOD from Bos taurus is available (PDB ID:2SOD), we were able to construct a 3D structure of Cu/Zn-SOD. The docking between Cu/Zn-SOD and TFE using DOCK6.3 was successful with a significant score (-11.52 kcal/mol). We found that the binding residues predicted to interact with TFE are THR37, ASP40, and GLU119. The docking simulation confirmed our experimental findings that the inactivation of Cu/Zn-SOD by TFE is not due to binding of TFE directly in the copper- and zinc-containing active site pocket.At a TFE concentration of less than 10%, the level of secondary structure increased slightly; however, as the concentration of TFE increased, the overall amount of secondary structure decreased gradually in a dose-dependent manner. Although TFE is known to stabilize helices within proteins, it actually decreased the extent of secondary structure in tyrosinase. Specifically, in the concentration range of 15% to 25% TFE, the spectrum of secondary structural changes decreased significantly, while at higher concentrations (25% to 35%) the spectrum of secondary structural changes increased. To elucidate the relationship betweep secondary structure changes and tyrosinase activity, we assayed the L-DOPA oxidase activity of tyrosinase in the presence of TFE. At concentrations of TFE less than 10%, tyrosinase activity was sustained, and even slightly activated.When TFE concentrations were higher than 10%, however, tyrosinase activity gradually decreased in a dose-dependent manner (IC50=21% or 2.9 M). To assay the reversibility of TFE-mediated modification, plots of the remaining activity versus [E] were applied. The results showed straight lines passing through the origin, confirming that the inhibition by TFE was reversible. To evaluate the mode of inhibition, Lineweaver-Burk plot analysis was performed. The apparent V max and K m both appeared to change simultaneously, and a secondary replot of slope versus [I] yielded a parabolic curve indicating that TFE induced a parabolic mixed-type inhibition. The K1 value was calculated as 0.5±0.096 M (3.63±0.7%) and the value of β was calculated as 1.09.To evaluate the inactivation kinetics and rate constants, time-interval measurements were performed. Enzyme activity was not inactivated in the presence of 10% TFE, and only when TFE was higher than 20% did activity gradually decrease in a time-dependent manner. This result is entirely consistent with the data presented, where the equilibrium state of the enzyme activity was not inactivated at less than 10% TFE. Subsequent kinetic analyses using semi-logarithmic plots showed biphasic inactivation, with fast (k1) and slow (k 2) aspects, where a monophasic process developed into a biphasic process as the concentration of TFE increased; the inhibition followed first-order kinetics. The microscopic inactivation rate constants: inactivation occurred as a result of changes in the transition free-energy energy (ΔΔG°’),which decreased in a TFE concentration-dependent manner. Tertiary structural changes of tyrosinase in the presence of TFE were also measured. We found that TFE induced a change in the intrinsic fluorescence spectra of tyrosinase, which gradually decreased via a red-shift wavelength effect. A plot of maximum peak wavelength versus the concentration of TFE revealed a sigmoid relationship and indicated that TFE induces tertiary structural disruption of tyrosinase. We also monitored the ANS-binding fluorescence changes; the ANS-fluorescence spectra were significantly increased by TFE, indicating that hydrophobic surfaces of tyrosinase were exposed by TFE. In addition, the increase was dose dependent, as observed from a secondary replot.Tertiary structural changes resulting from modulation of the secondary structure modulation therefore appear to be directly associated with the observed changes in enzyme activity. Because the crystallographic structure of tyrosinase has not been elucidated, we selected template structures from the PDB entries lwxc, lxom,2oic, and 2oid that have sequence identities of 25%,29%,26%, and 25% to tyrosinase, respectively, to simulate the 3D structure of tyrosinase. In the predicted structure of tyrosinase, we used the binding pocket expanded to a size of 496 A3. The docking between tyrosinase and TFE was successful with significant scores (-2.25 kcal/mol by Autodock4 and-14.36 kcal/mol by Dock6). Using Autodock4 and Dock6, we searched for TFE binding residues of tyrosinase that were close in distance. We found that the most important expected binding residues interacting with TFE were PHE170, THR175, VAL177, GLY251, PHE261, and ASP536 according to Autodock4, and GLU250 and ASP536 according to Dock6; both programs identified ASP536. The docking simulation provided supported for the slope-parabolic mixed-type inhibition observed, as this type of inhibition is generally observed when there are multiple binding sites for an inhibitor. The mechanism of Cu/Zn-SOD catalysis is therefore of interest, as are the folding dynamics of this protein. Little is known about the tertiary structure and the structural integrity of the active site of Cu/Zn-SOD. Therefore, we investigated the effects of trifluoroethanol (TFE) on the structure and function of Cu/Zn-SOD. TFE is a reagent that is widely used as a structure-inducing cosolvent. It has been used as a cosolvent to investigate proteins and peptides for more than four decades since circular dichroism (CD) and nuclear magnetic resonance (NMR) studies revealed that the presence of TFE increases the quantity of a-helices and P-sheets in peptides in TFE-water mixtures. TFE is therefore considered to be a stabilizer for most enzymes; however, TFE denatures some enzymes by disrupting their tertiary structures, resulting in the loss of activity. We investigated how TFE modifies the secondary and tertiary structure^of SOD and affects its activity.We propose an inhibitory mechanism for the effect of TFE on Cu/Zn-SOD based on inhibition kinetics and computational prediction results obtained in this study. Our results indicate that TFE-induced inactivation of Cu/Zn-SOD is due to conformational changes in the protein rather than disruption of the active site of the enzyme by chelating agents, we propose a new approach to tyrosinase inhibition:modulation of secondary and tertiary structures of tyrosinase directly involved with catalysis. We simulated the 3D structure of tyrosinase, evaluated its docking behavior with TFE, and investigated its putative binding residues (ASP169, ALA171, TRP173, PHE261, and ASP536 from Autodock4; PHE170 and TRP173 from DOCK6). Our results provide insight into tyrosinase inhibition kinetics and the conformational changes induced by TFE.Conclusion1) TFE solvent denatured Cu/Zn-SOD.2) The active site of Cu/Zn-SOD is very stable and more resistant to TFE than the overall structure.3) Low concentrations of TFE induced overall structural changes, but the active site still remained intact.4) TFE binds to Cu/Zn-SOD near but not in the active site.5) TFE does not directly compete with the substrate, but alters the tertiary structure of Cu/Zn-SOD.6) TFE ligand binding to tyrosinase causes a complex type of inhibition of enzymatic activity;7) Secondary structural changes of tyrosinase result in the exposure of hydrophobic surfaces;8) The putative binding residues for TFE are predicted by computational docking simulation;TFE inactivation of tyrosinase and SOD represents a novel strategy for inactivating tyrosinase and developing inhibitors. |
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