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.     

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.     

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 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.     

Hysteresis of mushroom tyrosinase: Lag period of cresolase activity

Mushroom tyrosinase presents a lag period in the expression of its cresolase activity depending on enzyme and substrate concentration in the reaction m     

Inhibition of the catecholase and cresolase activity of mushroom tyrosinase by azide

The inhibition of mushroom tyrosinase by azide is examined as a function of the concentrations of l-tyrosine, l-3,4-dihydroxyphenylalanine (l-Dopa), and oxygen at pH 5.6 and 7.0. Mixed inhibition is observed with respect to l-tyrosine, l-Dopa, and oxygen. The data are interpreted in terms of azide combining with both the oxidized and reduced forms of the enzyme. A scheme is presented for the catecholase and cresolase reactions which explains the results of azide inhibition and also the effect of other inhibitors which complex with the copper of tyrosinase. Double-reciprocal plots of oxygen variation with l-tyrosine as the fixed substrate are nonlinear above about 500 μm oxygen. When l-Dopa is the fixed substrate, no curvature is observed. These results could be explained in terms of negative cooperativity or the presence of two kinetically distinct enzyme forms having different K_m values for oxygen. Although the kinetic data do not permit a choice between the two possibilities, the occurrence in all tyrosinase preparations of two forms, resting, bicupric enzyme and “intrinsic oxytyrosinase,” lends support to the latter suggestion.     

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.     

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.     

Inactivation kinetics of mushroom tyrosinase by cetylpyridinium chloride

Cetylpyridinium chloride (CPC) was found to inactivate tyrosinase from mushroom (Agaricus bisporus). CPC can bind to the enzyme molecule and induce the enzyme conformation changes. The fluorescence intensity (at 338.4 nm) of the enzyme decreased distinctly with increasing CPC concentrations, and a new little fluorescence emission peak appeared near 372 nm. The inactivation of the enzyme by CPC had first been studied by using the kinetic method of the substrate reaction described by Tsou. The results showed that the enzyme was inactivated by a complex mechanism that had not been previously identified. The enzyme first quickly binds with CPC reversibly and then undergoes a slow irreversible inactivation. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is a saturated trend being independent of CPC concentration if the concentration is sufficiently high. The micro rate constants of inactivation and the association constant were determined.     

Inhibition of mushroom tyrosinase by tropolone

Tropolone inhibits both mono- and o-dihydroxyphenolase activity of mushroom tyrosinase. Most of the inhibition exerted by tropolone was reversed by dialysis or by excess CU²⁺. The data indicate that tropolone and o-dihydroxyphenols compete for binding to the copper at the active site of the enzyme. Comparison between the effectiveness of various copper chelators showed that tropolone is one of the most potent inhibitors of mushroom tyrosinase; 50% inhibition was observed with 0.4 × 10^?6 M tropolone.     

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.     

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。     

Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase

The effects of fluorobenzaldehydes (2-,3- and 4-fluorobenzaldehyde) on the activity of mushroom tyrosinase have been studied. The results show that fluorobenzaldehydes can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition is reversible. The IC₅₀ values were estimated as 1.62 mM, 1.06 mM and 0.16 mM for diphenolase activity and as 1.35 mM, 1.18 mM and 1.05 mM for monophenolase activity, respectively. The lag time of the monophenolase was obviously lengthened by these three fluorobenzaldehydes. When the concentration of inhibitors reached 2.0 mM, the lag time was lengthened from 33 s to 142 s, 168 s and 190 s, respectively. Kinetic analyses show that the inhibition mechanism of 2-fluorobenzaldehyde on the diphenolase was competitive inhibition of the diphenolase activity, and that of 3-fluorobenzaldehyde and 4-fluorobenzaldehyde were of a mixed-type. The inhibition constants for these three fluorobenzaldehydes on the diphenolase were determined and compared.     

Inhibitory effects of some flavonoids on the activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones, and then forms brown or black pigments. In the present study, the effects of some flavonoids on the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that flavonoids can lead to reversible inhibition of the enzyme. A kinetic analysis showed that the flavonols are competitive inhibitors, whereas luteolin is an uncompetitive inhibitor. The rank order of inhibition was: quercetin > galangin > morin; fisetin > 3,7,4"-trihydroxyflavone; luteolin > apigenin > chrysin.     

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.     

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.     

Inhibitory effects of cis- and trans-isomers of 3,5-dihydroxystilbene on the activity of mushroom tyrosinase

The effects of cis- and trans-isomers of 3,5-dihydroxystilbene on the activity of mushroom tyrosinase have been studied. The results show that both cis- and trans-isomers of 3,5-dihydroxystilbene can inhibit the diphenolase activity of the enzyme and the inhibition type was reversible. The IC(50) values were estimated as 0.405+/-0.013 and 0.705+/-0.017 mM, respectively. Kinetic analysis showed that the inhibition of cis-3,5-dihydroxystilbene and trans-3,5-dihydroxystilbene on the diphenolase activity of the enzyme belonged to competitive type, and the inhibition constants (K(I)) were determined to be 0.232+/-0.015 and 0.395+/-0.020 mM, respectively. In this investigation, the inhibitory effects of cis-3,5-dihydroxystilbene and trans-3,5-dihydroxystilbene on the diphenolase activity of mushroom tyrosinase were compared. The inhibitory capacity of cis-isomer was stronger than that of corresponding trans-isomer. Nevertheless, the trans-3,5-dihydroxystilbene was used more frequently than its corresponding cis-form compound. This research may offer some references for designing and synthesizing some novel and effective tyrosinase inhibitors. Furthermore, it may improve the use of stilbenes on the field of food preservation and depigmentation.     

Effect of an abundant human skin melanosomal 66 kDa protein (MP 66) on m urine tyrosinase: Its physiological implications on melanogenesis

A single polypeptide protein (MP 66) of molecular weight 66 kDa purified to homogeneity from melanosomes of normal human skin epidermal melanocytes, was partially characterized. The isoelectric point of MP 66 is in the range of 7.3 to 7.6. This protein, which was shown to inhibit partially purified human skin tyrosinase activity at pH 6.8, also inhibits murine tyrosinase at pH 6.8. However, at pH 5.0, it stimulates murine tyrosinase activity. The physiological implications of these results are discussed.     

Amide derivatives with pyrazole carboxylic acids of 5-amino-1,3,4-thiadiazole 2-sulfonamide as new carbonic anhydrase inhibitors: Synthesis and investigation of inhibitory effects

Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide were synthesized from 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride. Carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from human erythrocyte cells by the affinity chromatography method. The inhibitory effects of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 and new synthesized amides on these isozymes have been studied in vitro. The I₅₀ concentrations (the concentration of inhibitor producing a 50% inhibition of CA activity) against hydratase activity ranged from 1.2 to 2.2 nM for hCA-I and from 0.4 to 2 nM for hCA-II. The I₅₀ values against esterase activity ranged from 1.4 to 8 nM for hCA-I and from 1.3 to 6 nM for hCA-II. The K_i values were observed between 8.2·10^{? 5} to 6.2·10^{? 4} M for hCA-I and between 2.9·10^{? 4} to 8.2·10^{? 4} M for hCA-II. The comparison of new synthesized amides to 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 indicated that the new synthesized compounds (18–23) inhibit CA activity more potently than the parent compounds.     

Inhibitory effects on mushroom tyrosinase by flavones from the stem barks of Morus lhou (S.) Koidz

Five flavones displaying tyrosinase inhibitory activity were isolated from the stem barks of Morus lhou (S.) Koidz., a cultivated edible plant. The isolated compounds were identified as mormin (1), cyclomorusin (2), morusin (3), kuwanon C (4), and norartocarpetin (5). Mormin (1) was characterized as a new flavone possesing a 3-hydroxymethyl-2-butenyl at C-3. The inhibitory potencies of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC₅₀ values of compounds 1–5 for monophenolase activity were determined to be 0.088, 0.092, 0.250, 0.135 mM, and 1.2 μM, respectively. Mormin (1), cyclomorusin (2), kuwanon C (4) and norartocarpetin (5) exhibited competitive inhibition characteristics. Interestingly norartocarpetin (5) showed a time–dependent inhibition against oxidation of l–tyrosine: it also operated under the enzyme isomerization model (k₅ = 0.8424 min^{? 1}, k₆ = 0.0576 min^{? 1}, = 1.354 μM).