The effect of thiobarbituric acid on tyrosinase: inhibition kinetics and computational simulation

Tyrosinase plays various roles in organisms and much research has focused on the regulation of tyrosinase activity. We studied the inhibitory effect of thiobarbituric acid (TBA) on tyrosinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K(i) 5 14.0 ± 8.5 mM and IC?? 5 8.0 ± 1.0 mM). Intrinsic and ANS-binding fluorescences studies were also performed to gain more information regarding the binding mechanism. The results showed that no tertiary structural changes were obviously observed. For further insight, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and TBA. The docking simulation was successful with significant scores (binding energy for AutoDock4: -5.52 kcal/mol) and suggested that TBA was located in the active site. The 11 ns molecular dynamics simulation convinced that the four HIS residues (residue numbers: 57, 90, 250, and 282) were commonly responsible for the interaction with TBA. Our results provide a new inhibition strategy that works using an antioxidant rather than targeting the copper ions within the tyrosinase active site.     

Effects of isorhamnetin on tyrosinase: inhibition kinetics and computational simulation

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at Ki=0.235±0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: -32.58 kcal/mol, for AutoDock4.2: -5.66 kcal/mol, and for Fred2.2: -48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.     

The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (-1.89?kcal/mol) and the hexamer (-1.97?kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.     

The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (?1.89?kcal/mol) and the hexamer (?1.97?kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.     

Effect of Cl- on tyrosinase: complex inhibition kinetics and biochemical implication

Tyrosinase plays a core role in melanogenesis of the various organisms. Therefore, the regulation of the tyrosinase activity is directly related with melanin synthesis. In this study, we investigated the Cl(-)-induced inhibition of human tyrosinase and the potent role of Cl(-) as a negative regulator in melanogenesis. For the inhibition kinetic studies, human tyrosinase was differently prepared from the TXM13 melanotic cells as well as from cells that had undergone gene transfection. We found that Cl(-) inhibited tyrosinase in a slope-parabolic competitive manner and tyrosinase gene transfection into HEK293 cell significantly down-regulated the expression levels of solute carrier family 12, member 4 (potassium/chloride transporters, SLC12A7) and solute carrier family 12, member 7 (potassium/chloride transporters, SLC12A7), which are known to be Cl(-) transporters. From the results of the inhibition kinetic studies and the Cl(-) transporter expression level, we suggested that Cl(-) might act as a potent regulatory factor in melanogenesis. It is worth notice that a high content of Cl(-) exists physiologically and tyrosinase reacts sensitively to Cl- in a complex interaction manner.     

Effects of piperonylic acid on tyrosinase: Mixed-type inhibition kinetics and computational simulations

Piperonylic acid is a natural molecule with a benzoic acid group and high antioxidant capacity. Based on its aromatic acid structure and antioxidant properties, we studied the effects of piperonylic acid on tyrosinase by the analysis of its inhibitory kinetics and computational simulations. Piperonylic acid reversibly inhibited tyrosinase through a mixed-type inhibitory mechanism. The time courses of the tyrosinase inhibition showed that piperonylic acid binds to tyrosinase very quickly and the inactivation processes follow first-order kinetics. The continuous substrate reactions indicated that piperonylic acid induced a tight-binding inhibition and the substrate can promote the inactivation process. The ANS-binding fluorescence of tyrosinase suggested that piperonylic acid did not detectably disrupt the tertiary structure of the enzyme. The results of the computational docking and molecular dynamics simulations showed that piperonylic acid closely interacts with three residues and it might block the active site of tyrosinase.     

Reversible inhibition of protein splicing by zinc ion

Protein splicing involves the self-catalyzed excision of a protein-splicing element, the intein, from flanking polypeptides, the exteins, which are concomitantly joined by a peptide bond. Taking advantage of recently developed in vitro systems in which protein splicing occurs in trans to assay for protein-splicing inhibitors, we discovered that low concentrations of Zn(2+) inhibited splicing mediated both by the RecA intein from Mycobacterium tuberculosis and by the naturally split DnaE intein from Synechocystis sp. PCC6803. Inhibition by Zn(2+) was also observed with a cis-splicing system involving the RecA intein. In all experimental systems used, inhibition by Zn(2+) could be completely reversed by the addition of EDTA. Zinc ion also inhibited hydroxylamine-dependent N-terminal cleavage of the RecA intein. All other divalent transition metal ions tested were less effective as inhibitors than Zn(2+). The reversible inhibition by Zn(2+) should be useful in studies of the mechanism of protein splicing and allow structural studies of unmodified protein-splicing precursors.     

Effect of hesperetin on tyrosinase: inhibition kinetics integrated computational simulation study

Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K_i = 4.03 ± 0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: −34.41 kcal/mol and for AutoDock4.2: −5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms.     

The influence of hydroquinone on tyrosinase kinetics

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.     

The effect of histidine residue modification on tyrosinase activity and conformation: inhibition kinetics and computational prediction

We found that the histidine chemical modification of tyrosinase conspicuously inactivated enzyme activity. The substrate reactions with diethylpyridinecarbamate showed slow-binding inhibition kinetics (K(I) = 0.24 +/- 0.03 mM). Bromoacetate, as another histidine modifier, was also applied in order to study inhibition kinetics. The bromoacetate directly induced the exposures of hydrophobic surfaces following by complete inactivation via ligand binding. For further insights, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and diethylpyridinecarbamate. The docking simulation was shown to the significant binding energy scores (-3.77 kcal/mol by AutoDock4 and -25.26 kcal/mol by Dock6). The computational prediction was informative to elucidate the role of free histidine residues at the active site, which are related to substrate accessibility during tyrosinase catalysis.     

Synergistic inhibition of carboxypeptidase A by zinc ion and imidazole

Zinc ion in solution yields a 560-fold enhancement in the kinetic inhibition of carboxypeptidase A by the simple heterocycle imidazole, behavior attributed to formation of a ternary complex of the three species. However, the effect is partially negated by formation of less-inhibitory Zn2+(C3H4N2)2-4 coordination complexes, providing for the enzyme an anomalous profile of catalytic rate versus imidazole concentration.     

Effect of zinc ion on cadmium-induced auditory changes

Cadmium, which has adverse effects on many physiological systems, is an important environmental pollutant. Our previous experimental study showed that cadmium also has a dose-dependent deleterious effect on the auditory system in rats. Because zinc reverses cadmium cytotoxicity in many systems, we investigated the possible preventive effect of a zincenriched diet given isochronally on cadmium-induced hearing loss in rats. Fifty-four male rats were divided into three equal groups. Control rats were fed normal rat food and tap water, whereas the cadmium group was subjected to 15 ppm cadmium-containing water as CdCl₂. The third group received 15 ppm CdCl₂ and food enriched with 200 ppm zinc as ZnSO₄ for 30 d. On d 30, eight animals from each group were used for the measurement of kidney functions. In the remaining animals, hearing functions were measured by auditory brainstem response and distortion product otoacoustic emission. Blood cadmium increased from 1.87±1.69 to 6.08±2.62 μg/dL and elevated cadmium contents of ear ossicles and kidney cortex were associated with a decreased glomerular filtration rate in rats subjected to high cadmium. A zinc-enriched diet obviously reduced cadmium accumulation in the kidney and prevented the nephrotoxicity. Our data indicated that cadmium-induced ototoxicity seems to be partially zinc preventable and zinc addition to diet without altering cadmium content in ear ossicles may help to prevent cadmium-induced hearing loss.     

The inhibition of tyrosinase by pyridinones.

3-Hydroxypyridine-4-ones have potential as orally active chelators of iron(III) and therefore may find application in the treatment of thalassaemia. An undesirable feature of these molecules is that they inhibit tyrosinase. We have established that alkyl substitution at position 2 in the aromatic ring minimizes interaction with tyrosinase and does so without appreciably influencing the affinity for iron(III).     

Inhibition of mushroom tyrosinase by a newly synthesized ligand: inhibition kinetics and computational simulations

Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl)phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competitively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6?μM, respectively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts.     

Transient kinetics of thermodynamic buffering

The transient response of mitochondrial ATP production towards perturbations was studied by analyzing the trajectories leading from arbitrary initial conditions of the adenine nucleotide pool to the final steady state. These trajectories were calculated from differential equations based on linear relations between flows and thermodynamic forces of the adenylate kinase system including oxidative phosphorylation. The motion of the system along the trajectories consists of two phases: (1) a rapid phase leading from initial states to a common relaxation curve; and (2) a slow phase leading along the relaxation curve to the final steady state. The first phase corresponds to a motion close to the loci of constant adenylic energy charge. In line with this observation is the finding that the energy charge is a constant of motion of the adenylate kinase reaction. The second phase corresponds to a motion along a relaxation curve characterized by minimal Lyapunov exponents in the concentration space of the adenine nucleotides. Thus, both phases of the transient kinetics can be approximated in terms of thermodynamic functions to a high degree of precision. Incubations with isolated rat liver mitochondria were in excellent agreement with the theoretical predictions. In summary, these studies show that the adenylate kinase system not only optimizes the efficiency of oxidative phosphorylation through thermodynamic buffering but, in addition, also deeply influences the transient response of the whole system.     

Antioxidation and tyrosinase inhibition of polyphenolic curcumin analogs

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver-Burk plots, revealed that compounds B(2) and C(2) bearing o-diphenols were non-competitive inhibitors, while compounds B(11) and C(11) bearing m-diphenols were competitive inhibitors. In particular, representative compounds C(2) and B(11) showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.     

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.     

Magnesium ion dependent equilibria, kinetics, and thermodynamic parameters of Artemia ribosome dissociation and subunit association

The influence of magnesium ion concentration on the equilibrium and kinetics of Artemia ribosome dissociation and subunit association has been studied by laser light scattering. Ribosomal aggregation was found to be reduced by addition of 0.1-0.05 mM spermidine and KCl concentrations of 100 mM. The ribosomes were found to be stable at low [Mg2+], and the curves obtained for ribosome-subunit equilibrium were independent of the direction and origin of the magnesium ion titration. Thermodynamic parameters were obtained from the temperature-dependent equilibria and have been compared to those of wheat germ and Escherichia coli type A ribosomes. The entropy term calculated for the association of 40S and 60S subunits is small, and the reaction is exothermic. The entropy term is negative, favoring subunit dissociation, and contributes less to the free energy than the enthalpy term. Rate constants for ribosome dissociation and subunit association have been determined. The reaction curves gave no evidence for sequential processes and were homogeneous.