The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C₃H₇OCS₂Na (I), C₄H₉OCS₂Na (II) and C₅H₁₁OCS₂Na (III), were synthesized, by the reaction between CS₂ with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K_i values of 9.8, 7.2 and 6.1 μM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S^? ) of the anion xanthate, which leads to similar values of K_i for all three xanthates. Different K_i values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C₄H₉NHCS₂Na (I), C₆H₁₃NHCS₂Na (II) and C₈H₁₇NHCS₂Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver–Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to K_i values of 0.8, 1.0 and 1.8 μM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different K_i values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C4H9NHCS2Na (I), C6H13NHCS2Na (II) and C8H17NHCS2Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to Ki values of 0.8, 1.0 and 1.8 microM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different Ki values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

氨基葡萄糖对酪氨酸酶的抑制作用

通过对酪氨酸酶催化底物L-DOPA反应速率的观察测定,研究了氨基葡萄糖（G-NH2）对酪氨酸酶的抑制作用。在反应液中加入50μL浓度为2.2 mg/mL G-NH2时（体系中G-NH2终浓度为36μg/mL）,酶抑制率为50%。GNH2对酪氨酸酶的抑制作用是个复杂的过程,酶反应呈先促进后抑制。分析酶抑制曲线Lineweaver-Burk双倒数图,得出G-NH2为混合抑制剂,进一步研究发现多巴醌生产量会减少,抑制类型是不可逆抑制。     

The inhibitory effect of ethylenediamine on mushroom tyrosinase

The inhibitory effect of ethylenediamine on both activities of mushroom tyrosinase (MT) at 20 °C in a 10 mM phosphate buffer solution (pH 6.8), was studied. L-DOPA and L-tyrosine were used as substrates of catecholase and cresolase activities, respectively. The results showed that ethylenediamine competitively inhibits both activities of the enzyme with inhibition constants (K(i)) of 0.18±0.05 and 0.14±0.01 μM for catecholase and cresolase respectively, which are lower than the reported values for other MT inhibitors. For further insight a docking study between tyrosinase and ethylenediamine was performed. The docking simulation showed that ethylenediamine binds in the active site of the enzyme near the Cu atoms and makes 3 hydrogen bonds with two histidine residues of active site.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30 degrees C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K(i)) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K(i) value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30 degrees C at both pHs. Moreover, the effect of temperature on K(i) value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30°C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K_i) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K_i value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30°C at both pHs. Moreover, the effect of temperature on K_i value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

Molecular inhibitory mechanism of dihydromyricetin on mushroom tyrosinase

Tyrosinase is the rate-limiting enzyme for controlling the production of melanin in the human body, and overproduction of melanin can lead to a variety of skin disorders. In this paper, the inhibitory kinetics of Dihydromyricetin (DHM) on tyrosinase and their binding mechanism were determined using spectroscopy, molecular docking, antioxidant assays, and chromatography. The spectroscopic results indicate that DHM reversibly inhibits tyrosinase in a mixed-type manner through a multiphase kinetic process with the IC₅₀ of 849.88 μM. It is shown that DHM has a strong ability to quench the intrinsic fluorescence of tyrosinase mainly through a static quenching procedure, suggesting that a stable DHM–tyrosinase complex is generated. Molecular docking results suggest that the dominant conformation of DHM does not directly bind to the active site of tyrosinase. Moreover, the antioxidant assays demonstrate that DHM has powerful antioxidant and reducing capacity but does not have the ability to reduce dopachrome to L-DOPA. Interestingly, the results of spectroscopy and chromatography indicate that DHM is a substrate of tyrosinase but not a suicide substrate. The possible inhibitory mechanism is proposed, which will be helpful to design and search for tyrosinase inhibitors.     

Potent inhibitory effects of benzyl and p-xylidine-bis dithiocarbamate sodium salts on activities of mushroom tyrosinase

A novel monofunctional benzyldithiocarbamate, C₆H₅CH₂NHCSSNa (I), and a bifunctional p-xylidine-bis(dithiocarbamate), NaSSCNHCH₂C₆H₄CH₂NHCSSNa (II), as sodium salts, were synthesized by reaction between p-xylylenediamine or benzylamine with CS₂ in the presence of NaOH. They were characterized by spectroscopic techniques such as ¹H NMR, IR, and elemental analysis. These water-soluble compounds were examined for their inhibition of both activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. l-3,4- Dihydroxyphenylalanine (L-DOPA) and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic studies showed noncompetitive inhibition of I and mixed type inhibition of II on both activities of MT. The inhibition constant (K_I) of II was smaller than that of I. Raising the temperature from 27 to 37°C caused a decrease in K_I values of I and an increase in values of II. The binding process for inhibition of I was only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic; meanwhile, the electrostatic interaction can be important for the inhibition of II due to the enthalpy driven binding process. Fluorescence studies showed a decrease of emission intensity without a shift of emission maximum in the presence of different concentrations of compounds. An extrinsic fluorescence study did not show any considerable change of the tertiary structure of MT. Probably, the conformation of inhibitor-bound MT is stable and inflexible compared with uninhibited MT.     

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase

A novel tyrosinase inhibitor, an arbutin derivative having undecylenic acid at the 6-position of its glucose moiety, was enzymatically synthesized. Its inhibitory activity was studied in vitro by using catechol and phenol as substrates. The IC₅₀ value of the arbutin ester on tyrosinase using catechol (4 × 10⁻⁴ M) was 1% of that when arbutin (4 × 10⁻² M) was used. Using phenol, IC₅₀ of the arbutin ester (3 × 10⁻⁴ M) as substrate was 10% of that of arbutin (3 × 10⁻³ M). These results suggest that the arbutin ester inhibits the latter part of the tyrosinase reaction, which consists of hydroxylation and oxidation.     

Multiple effect of hydroxylamine on mushroom tyrosinase

Mushroom tyrosinase is affected by hydroxylamine (NH₂OH) in several ways. At relatively low concentrations (up to 33 mM) NH₂OH shortens the lag period of tyrosine hydroxylation. The o-dihydroxyphenolase activity of mushroom tyrosinase is slightly stimulated by short exposure to relatively low concentrations ofNH₂OH (1.5 mM). Relatively high concentrations ofNH₂OH (above 20 mM) inhibit the o-dihydroxyphenolase activity of the enzyme and lowers the extent of final pigment production. Preincubation of mushroom tyrosinase with different concentrations ofNH₂OH for different times results in the inactivation of the enzyme. The rate of inactivation occurred much faster under anaerobic than under aerobic conditions. It was also found that NH₂OH changes the spectra of o-quinones prepared chemically or of products formed during the oxidation of o-dihydroxyphenols by mushroom tyrosinase. These spectral changes were attributed to the formation of oximes (mono- or dioximes) as a result of an interaction between o-quinones and NH₂OH. The apparent inhibition exerted by NH₂OH on the o-dihydroxyphenolase activity of mushroom tyrosinase is, in part, due to spectral changes in pigmented product formation and, in part, due to the inactivation of the enzyme by NH₂OH.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity

Background

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.

Methodology/Principal Findings

Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe₃O₄ were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.

Conclusions/Significance

The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.     

Depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities

We synthesized benzoate ester derivatives of kojic acid with and without adamantane moiety. Benzoate derivatives 2a-e that did not contain an adamantane moiety showed potent tyrosinase inhibitory activities. However, depigmenting activity was not noted in a cell-based assay. Contrasting results were obtained for benzoate derivatives (3a-e) containing an adamantane moiety. Compounds 3a-e showed potent depigmenting activities without tyrosinase inhibitory activities. To the best of our knowledge, this is the first study showing the depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities.     

A quantitative method for the determination of the activities of mushroom tyrosinase

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional enzyme, mushroom tyrosinase (o-diphenol: O₂ oxido-reductose, EC 1.10.3.1) has been developed. The spectrophotometric method utilizes p-cresol and 4-methyl catechol as substrates at pH 4.8. By maintaining this low pH value, the rates of the nonenzymic reactions are negligible during the course of the assay. Preliminary analysis of the rates of enzyme-catalyzed reactions gave typical results for both substrates: Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentration. Tyrosinase preparations judged to be as pure as those previously reported could be assayed to enzyme concentrations as low as 1 mg/liter with p-cresol while catechol allowed lower concentrations to be assayed (0.3 mg/liter). The precise specific activities towards p-cresol and 4-methyl catechol were found to vary between enzyme solutions and were used to characterize enzyme preparations.     

Irreversibly inhibitory kinetics of 3,5-dihydroxyphenyl decanoate on mushroom (Agaricus bisporus) tyrosinase

3,5-Dihydroxyphenyl decanoate (DPD) is found to inhibit the diphenolase activity of tyrosinase from mushroom (Agaricus bisporus). The effects of DPD on the diphenolase activity of mushroom tyrosinase have been studied. The results show that the enzyme activity decreases very slowly with an increase in DPD concentrations at lower concentrations of DPD (between 5 and 60 microM). But at higher concentrations of DPD, DPD can strongly inhibit the diphenolase activity of the enzyme and the inhibition is irreversible. The IC50 value was estimated to be 96.5 microM. The inhibition mechanism of DPD has been investigated and the results show that DPD can bind to the free enzyme molecule and enzyme-substrate complex and lose the enzyme activity completely. The inhibition kinetics has been studied in detail by using the kinetic method of the substrate reaction described by Tsou. The microscopic rate constants of the enzyme inhibited by DPD at higher concentrations have been determined.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Effect of alkyl chain length on sphingosine kinase 2 selectivity

The conversion of sphingosine to sphingosine-1-phosphate is catalyzed by sphingosine kinase (SphK), which has been implicated in disease states such as cancer and fibrosis. Because SphK exists as two different isoforms, SphK1 and SphK2, understanding the physiological function of each isoenzyme is important. Of the two isoenzymes, SphK2 is significantly less understood, which is evident by the lack of selective small molecule inhibitors. Building on our initial work that focused on the structure–activity relationship study on an FTY720-derived cylohexylamine scaffold, we report that varying the alkyl chain length on the hydrophobic tail can impart selectivity toward SphK2 over SphK1.