The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation.

Oxaloacetic acid (OA) is naturally found in organisms and well known as an intermediate of citric acid cycle producing ATP. We evaluated the effects of OA on tyrosinase activity and structure via integrating methods of enzyme kinetics and computational simulations. OA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type (IC50=17.5±0.5mM and Ki=6.03±1.36mM). Kinetic measurements by real-time interval assay showed that OA induced multi-phasic inactivation process composing with fast (k1) and slow (k2) phases. Spectrofluorimetry studies showed that OA mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational docking simulations further revealed that OA could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS259, HIS263, and VAL283. Our study provides insight into the mechanism by which energy producing intermediate such as OA inhibit tyrosinase and OA is a potential natural anti-pigmentation agent.

Effect of Cd2+ on tyrosinase: Integration of inhibition kinetics with computational simulation.

Cadmium ions (Cd2+) are a widespread and easily absorbed toxin to both humans and animals that can be spread via food, water, and air pollution. Tyrosinase (EC 1.14.18.1) is a multifunctional copper-containing enzyme that is ubiquitously expressed in animals and plays a critical role in melanin production. We evaluated the toxic effects of Cd2+ on tyrosinase activity and conformation by measuring kinetics and computationally simulating the interactions. We found Cd2+ to be a slope-hyperbolic noncompetitive-inhibition reversible inhibitor of tyrosinase, with an IC50 of 2.92±0.16mM and Ki of 0.23±0.02mM. Spectrofluorimetric measurements of intrinsic and ANS-binding fluorescence showed that Cd2+ did not induce significant changes to tyrosinase overall or to its regional active site conformations. Cd2+ showed its inactivation effect not by modulating apparent structural changes to tyrosinase, but by occupying binding sites. To gain further insight into the Cd2+/tyrosinase interaction, we performed computational docking and molecular dynamics simulations. The results consistently indicated that Cd2+ can interact with several residues near the tyrosinase active site, primarily HIS85 and ASN260. Our study provides insight into the mechanism of the toxic effects Cd2+ has on tyrosinase, which could affect the normal pigmentation pathway in animals.

The effect of alpha-ketoglutaric acid on tyrosinase activity and conformation: Kinetics and molecular dynamics simulation study.

Alpha-ketoglutaric acid (AKG) is naturally found in organisms and is a well-known intermediate in the production of ATP or GTP in the Krebs cycle. We elucidated the effects of AKG on tyrosinase activity and conformation via methods of inhibition kinetics integrated with molecular dynamics (MD) simulations. AKG was found to be a reversible inhibitor of tyrosinase (IC50=15±0.5mM) and induced parabolic slope mixed-type inhibition. Based on our newly established equation, the dissociation constant (Kislope) was determined to be 7.93±0.31mM. The spectrofluorimetry studies showed that AKG mainly induced regional changes in the active site of tyrosinase, which reflects the flexibility of the active site. The computational docking and molecular dynamics (MD) simulations further demonstrated that AKG could interact with several residues near the substrate-binding site located in the tyrosinase active site pocket. Our study provides insight into the mechanism by which energy-producing intermediates such as AKG inhibit tyrosinase through its ketone groups. Also, AKG could be a potential natural antipigmentation agent due to its non-toxic property.

Inhibition of tyrosinase by fumaric acid: Integration of inhibition kinetics with computational docking simulations.

Fumaric acid (FA), which is naturally found in organisms, is a well known intermediate of the citric acid cycle. We evaluated the effects of FA on tyrosinase activity and structure via enzyme kinetics and computational simulations. FA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type with IC50=13.7±0.25mM and Kislope=12.64±0.75mM. We newly established the equation for the dissociation constant (Kislope) for the parabolic inhibition type in this study. Kinetic measurements and spectrofluorimetry studies showed that FA induced regional changes in the active site of tyrosinase. One possible binding site for FA was identified under the condition without L-DOPA. The computational docking simulations further revealed that FA can interact with HIS263 and HIS85 at the active site. Furthermore, four important hydrogen bonds were found to be involved with the docking of FA on tyrosinase. Our study provides insight into the mechanism by which dicarboxylic acids such as FA inhibit tyrosinase. By inhibiting tyrosinase and its central role in pigment production, FA is a potential natural antipigmentation agent.

Effect of Cadmium Ion on alpha-Glucosidase: An Inhibition Kinetics and Molecular Dynamics Simulation Integration Study.

α-Glucosidase is a critical metabolic enzyme that produces glucose molecules by catalyzing carbohydrates. The aim of this study is to elucidate biological toxicity of Cd(2+) based on α-glucosidase activity and conformational changes. We studied Cd(2+)-mediated inactivation as well as conformational modulation of α-glucosidase by using kinetics coupled with simulation of molecular dynamics. The enzyme was significantly inactivated by Cd(2+) in a reversibly binding behavior, and Cd(2+) binding induced a non-competitive type of inhibition reaction (the K i was calculated as 0.3863 ± 0.033 mM). Cd(2+) also modulated regional denaturation of the active site pocket as well as overall partial tertiary structural change. In computational simulations using molecular dynamics, simulated introduction of Cd(2+) induced in a depletion of secondary structure by docking Cd(2+) near the saccharides degradation at the active site, suggesting that Cd(2+) modulating enzyme denaturation. The present study elucidated that the binding of Cd(2+) triggers conformational changes of α-glucosidase as well as inactivates catalytic function, and thus suggests an explanation of the deleterious effects of Cd(2+) on α-glucosidase.

Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.

Effects of L-malic acid on alpha-glucosidase: inhibition kinetics and computational molecular dynamics simulations.

The inhibitory effect of L-malic acid (MA) on alpha-glucosidase (EC 3.2.1.20) was investigated by combination study between inhibition kinetics and computational simulations. The results from the serial kinetics demonstrated that MA could directly inactivate the enzyme activity in a dose-dependent manner and a typical non-competitive type, as well as in a fast inactivate process without detectable time course. The tertiary conformation study with an application of spectrofluorimetry showed that MA modulated the tertiary structural conformation of alpha-glucosidase both on the overall and on regional active site pocket, which monitored by red-shift intrinsic fluorescence peak with decreases intensities, and the significant intensity increasing of 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence, respectively. To have more insight, we also adapted the computational molecular dynamics (MD) simulations. The results showed that MA was located in the entrance of active pocket for the catalytic reaction and blocked the passage of substrate. It confirmed that MA inhibits as a non-competitive type, not direct docking to the glucose binding site. Our study provides important molecular mechanisms to figure out alpha-glucosidase inhibition that might associate to development of type 2 diabetes mellitus drug.

Effect of Ca(2+) on the activity and structure of α-glucosidase: inhibition kinetics and molecular dynamics simulations.

Understanding the mechanism of inhibition of α-glucosidase (EC 3.2.1.20) is clinically important because of the involvement of this enzyme in type 2 diabetes mellitus. In this study, we conducted inhibition kinetics of α-glucosidase with Ca(2+) and 10-ns molecular dynamics simulations. We found that direct binding of Ca(2+) to the enzyme induced structural changes and inhibited enzyme activity. Ca(2+) inhibited α-glucosidase in a mixed-type reaction (Ki = 27.0 ± 2.0 mM) and directly induced the unfolding of α-glucosidase, which resulted in the exposure of hydrophobic residues. The simulations suggest that thirteen Ca(2+) ions may interact with α-glucosidase residues and that the Ca(2+) binding sites are associated with the structural changes in α-glucosidase. Our study provides insight into the mechanism of the Ca(2+)-induced structural changes in α-glucosidase and the inhibition of ligand binding. These results suggest that Ca(2+) could act as a potent inhibitor of α-glucosidase for the treatment of type 2 diabetes mellitus.

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems.

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100 g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8 M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.

Trifluoroethanol-induced changes in activity and conformation of manganese-containing superoxide dismutase.

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, -9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.

Towards Al-induced manganese-containing superoxide dismutase inactivation and conformational changes: an integrating study with docking simulations.

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in skins exposed to oxygen. We studied the inhibitory effects of Al(3+) on the activity and conformation of manganese-containing SOD (Mn-SOD). Mn-SOD was significantly inactivated by Al(3+) in a dose-dependent manner. The kinetic studies showed that Al(3+) inactivated Mn-SOD follows the first-order reaction. Al(3+) increased the degree of secondary structure of Mn-SOD and also disrupted the tertiary structure of Mn-SOD, which directly resulted in enzyme inactivation. We further simulated the docking between Mn-SOD and Al(3+) (binding energy for Dock 6.3: -14.07 kcal/mol) and suggested that ASP152 and GLU157 residues were predicted to interact with Al(3+), which are not located in the Mn-contained active site. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of Al(3+) and allow us to describe a ligand binding via inhibition kinetics combined with the computational prediction.

Mixed-type inhibition of tyrosinase from Agaricus bisporus by terephthalic acid: computational simulations and kinetics.

Tyrosinase inhibition studies are needed due to the agricultural and medicinal applications. For probing effective inhibitors of tyrosinase, a combination of computational prediction and enzymatic assay via kinetics were important. We predicted the 3D structure of tyrosinase from Agaricus bisporus, used a docking algorithm to simulate binding between tyrosinase and terephthalic acid (TPA) and studied the reversible inhibition of tyrosinase by TPA. Simulation was successful (binding energies for Autodock4 = -1.54 and Fred2.0 = -3.19 kcal/mol), suggesting that TPA interacts with histidine residues that are known to bind with copper ions at the active site. TPA inhibited tyrosinase in a mixed-type manner with a K ( i ) = 11.01 ± 2.12 mM. Measurements of intrinsic and ANS-binding fluorescences showed that TPA induced no changes in tertiary structure. The present study suggested that the strategy of predicting tyrosinase inhibition based on hydroxyl groups and orientation may prove useful for screening of potential tyrosinase inhibitors.

Trehalose has a protective effect on human brain-type creatine kinase during thermal denaturation.

We investigated the effects of trehalose on thermal inactivation and aggregation of human brain-type creatine kinase (hBBCK) in this study. In the presence of 1.0 M trehalose, the midpoint temperature of thermal inactivation (T (m)) of hBBCK increased by 4.6 °C, and the activation energy (E (a)) for thermal inactivation increased from 29.7 to 41.1 kJ mol(-1). Intrinsic fluorescence spectra also showed an increase in the apparent transition temperature (T (1/2)) of hBBCK from 43.0 °C to 46.5 °C, 47.7 °C, and 49.9 °C in 0, 0.6, 0.8, and 1.2 M trehalose, respectively. In addition, trehalose significantly blocked the aggregation of hBBCK during thermal denaturation. Our results indicate that trehalose has potential applications as a thermal stabilizer and may aid in the folding of other enzymes in addition to hBBCK.

Tyrosinase inhibition by isophthalic acid: kinetics and computational simulation.

Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with K(i)=17.8 ± 1.8mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: -25.19 kcal/mol and for AutoDock4.2: -4.28 kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.

Trifluoroethanol-induced activity and structural changes in bos taurus copper- and zinc-containing superoxide dismutase.

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in organisms exposed to oxygen. Copper- and zinc-containing SOD (Cu/Zn-SOD) catalysis and the change in folding behavior of this enzyme in response to inactivators are therefore of interest. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of a Cu/Zn-SOD from Bos taurus. We found that TFE inactivated the enzyme and disrupted the tertiary and secondary structures of Cu/Zn-SOD. Kinetic studies showed that TFE-induced inactivation of Cu/Zn-SOD follows first-order reaction kinetics and that TFE binding sites are distinct from the copper- and zinc-containing active site. These structural changes occurred prior to enzyme activity loss. A computational docking simulation of Cu/Zn-SOD and TFE (binding energy of Dock 6.3: -11.52 kcal/mol) suggested that THR37, ASP40, and GLU119, which are located near the active site, interact with TFE. Evaluation of the ligand binding kinetics of Cu/Zn-SOD during unfolding in the presence of TFE combined with computational prediction allowed us to gain insight into the inactivation of Cu/Zn-SOD.

High-throughput integrated analyses for the tyrosinase-induced melanogenesis: microarray, proteomics and interactomics studies.

The tyrosinase gene was overexpressed in HEK293 cells, and then a DNA microarray and proteomic tools were applied to detect the dysregulated genes in highly pigmented cells. The candidate genes from the microarray were compared to the yeast two-hybridization results. Computational prediction via protein-protein interaction mapping suggested the existence of 66 hub genes in melanogenesis. Most importantly, RNA binding motif protein 9 is newly detected as a putative critical melanogenesis-associated gene in this study. The approach of combining the expression data analysis and predicted protein interaction partners performed in large scales can bring more reliable gene targets for understanding pigmentation.

The effect of trifluoroethanol on tyrosinase activity and conformation: inhibition kinetics and computational simulations.

We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of tyrosinase. TFE increased the degree of secondary structure of tyrosinase, which directly resulted in enzyme inactivation. A reciprocal study showed that TFE inhibited tyrosinase in a slope-parabolic mixed-type inhibition manner (K (I) = 0.5 +/- 0.096 M). Time-interval kinetic studies showed that the inhibition was best described as first order with biphasic processes. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescences were also measured to gain more insight into the supposed structural changes; these showed that TFE induced a conspicuous tertiary structural change in tyrosinase by exposing hydrophobic surfaces. We also predicted the tertiary structure of tyrosinase and simulated its docking with TFE. The docking simulation was successful with significant scores (binding energy for Autodock4 = -4.75 kcal/mol; for Dock6 = -23.07 kcal/mol) and suggested that the TRP173 residue was mainly responsible for the interaction with TFE. Our results provide insight into the structure of tyrosinase and allow us to describe a new inhibition strategy that works by inducing conformational changes rather than targeting the active site of the protein.

Structural analysis and inhibitory kinetics of brain type creatine kinase by sodium dodecyl sulfate.

The studies regarding the effect of sodium dodecyl sulfate (SDS) on enzyme activities and structures can provide a valuable insight into public health. We have predicted the 3D structure of the brain creatine kinase (CK-BB) with a high resolution and simulated the docking between CK-BB and SDS. The predicted structure had a root mean square deviation of 0.51 A. The docking between CK-BB and SDS was successful with significant scores (-4.67 kcal/mol, AutoDock4 and -48.32 kcal/mol, DOCK6). We have also investigated the inactivation by using SDS to study CK-BB's folding behaviors. The two-phase rate constants as a first-order reaction were measured during inactivation. SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner (K (i) = 1.22 mM). The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface. The methyl-beta-cyclodextrin was used to strip SDS from the enzyme molecule to reactivate. The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy, Gibbs free energy, and entropy were obtained as -13 + or - 7.0 MJ/mol, 8.39 kJ/mol, and -42.754 kJ/(K mol), respectively. Our study provides important structural information for CK-BB and its interaction with SDS with an insight on its folding and inhibition kinetics.