Zhi-Jiang Wang

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.

An integrated study of tyrosinase inhibition by rutin: progress using a computational simulation.

Abstract Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: −9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with Ki = 1.10 ± 0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent antipigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.

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

Abstract 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 tyro- sinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K i = 14.0 ± 8.5 mM and IC50 = 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 K i=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.

Computational Prediction of Protein-Protein Interactions of Human Tyrosinase

The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms.

A folding study of Antarctic krill (Euphausia superba) alkaline phosphatase using denaturants

To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions.

The effect of validamycin A on tyrosinase: inhibition kinetics and computational simulation.

In this study, we investigated validamycin A as a tyrosinase inhibitor based on its structural properties. We found that the reversible inhibition of tyrosinase by validamycin A occurred in a mixed-type manner with Ki=5.893±0.038mM, as determined by integrating kinetics studies and computational simulations. Time-interval tyrosinase studies showed that the inhibition followed first-order kinetics with two phases. Fluorescence measurements of ANS binding showed that validamycin A induced changes in the tertiary protein structure of tyrosinase. To obtain further insight, computational docking and molecular dynamics were applied, and the results indicated that HIS85, HIS244, GLU256, HIS259, and ASN260 of tyrosinase interacted with validamycin A. This strategy of predicting tyrosinase inhibition based on hydroxyl group numbers might be useful in the design and screening of potential tyrosinase inhibitors.

Folding studies on muscle type of creatine kinase from Pelodiscus sinensis

A folding study of creatine kinase from Pelodiscus sinensis has not yet been reported. To gain more insight into structural and folding mechanisms of P. sinensis CK (PSCK), denaturants such as SDS, guanidine HCl, and urea were applied in this study. We purified PSCK from the muscle of P. sinensis and conducted inhibition kinetics with structural unfolding studies under various conditions. The results revealed that PSCK was completely inactivated at 1.8 mM SDS, 1.05 M guanidine HCl, and 7.5 M urea. The kinetics via time-interval measurements showed that the inactivation by SDS, guanidine HCl, and urea were all first-order reactions with kinetic processes shifting from monophase to biphase at increasing concentrations. With respect to tertiary structural changes, PSCK was unfolded in different ways; SDS increased the hydrophobicity but retained the most tertiary structural conformation, while guanidine HCl and urea induced conspicuous changes in tertiary structures and initiated kinetic unfolding mechanisms. Our study provides information regarding PSCK and enhances our knowledge of the reptile-derived enzyme folding.

Purification, characterization, and unfolding studies of arginine kinase from Antarctic krill.

The regulation of enzymatic activity and unfolding studies of arginine kinase (AK) from various invertebrates have been the focus of investigation. To gain insight into the structural and folding mechanisms of AK from Euphausia superba (ESAK), we purified ESAK from muscle properly. The enzyme behaved as a monomeric protein with a molecular mass of about 40kDa and had pH and temperature optima of 8.0 and 30°C, respectively. The Km(Arg) and Km(ATP) for the synthesis of phosphoarginine were 0.30 and 0.47mM, respectively, and kcat/Km(Arg) was 282.7s(-1)/mM. A study of the inhibition kinetics of structural unfolding in the denaturant sodium dodecyl sulfate (SDS) was conducted. The results showed that ESAK was almost completely inactivated by 1.0mM SDS. The kinetics analyzed via time-interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to biphase as SDS concentrations increased. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence showed that SDS concentrations lower than 5mM did not induce conspicuous changes in tertiary structures, while higher concentrations of SDS exposed hydrophobic surfaces and induced conformational changes. These results confirmed that the active region of AK is more flexible than the overall enzyme molecule.