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.     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Inactivation of mushroom tyrosinase by hydrogen peroxide

Hydrogen peroxide (H₂O₂) inactivates mushroom tyrosinase in a biphasic manner, with the rate being faster in the first phase than in the second one. The inactivation of the enzyme is dependent on H₂O₂ concentration (in the range of 0.05–5.0 mM), but independent of the pH (in the range of 4.5–8.0). The rate of inactivation of mushroom tyrosinase by H₂O₂ is faster under anaerobic conditions (nitrogen) than under aerobic ones (air). Substrate analogues such as L-mimosine, L-phenylalanine, p-fluorophenylalanine and sodium benzoate protect the enzyme against inactivation by H₂O₂. Copper chelators such as tropolone and sodium azide also protect the enzyme. Under identical conditions, apotyrosinase is not inactivated by H₂O₂, unlike holotyrosinase. The inactivation of mushroom tyrosinase is not accelerated by an OH^?dot generating system (Fe²⁺-EDTA-H₂O₂) nor is it protected by OH^dot scavengers such as mannitol, urate, sodium formate and histidine. Exhaustive dialysis or incubation with catalase does not restore the activity of H₂O₂-inactivated enzyme. The data suggest that Cu²⁺ at the active site of mushroom tyrosinase is essential for the inactivation by H₂O₂. The inactivation does not occur via the OH^dot radical in the bulk phase but probably via an enzyme-bound OH^dot.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of 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 paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

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.     

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.     

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.     

Indirect inactivation of tyrosinase in its action on 4-tert-butylphenol

Abstract

Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates met and deoxytyrosinase enzymatic forms of tyrosinase. However, the monophenol 4-tert-butylphenol (TBF) protects the enzyme from this inactivation. Under aerobic conditions, the enzyme suffers suicide inactivation when it acts on TBC. We suggest that TBF does not directly cause the suicide inactivation of the enzyme in the hydroxylase activity, but that the o-diphenol, which is necessary for the system to reach the steady state, is responsible for the process. Therefore, monophenols do not induce the suicide inactivation of tyrosinase in its hydroxylase activity, and there is a great difference between the monophenols that give rise to unstable o-quinones such as L-tyrosine, which rapidly accumulate L-dopa in the medium and those like TBF, after oxidation, give rise to a very stable o-quinone.     

Dual effects of aliphatic carboxylic acids on cresolase and catecholase reactions of mushroom tyrosinase

Catecholase and cresolase activities of mushroom tyrosinase (MT) were studied in presence of some n-alkyl carboxylic acid derivatives. Catecholase activity of MT achieved its optimal activity in presence of 1.0, 1.25, 2.0, 2.2 and 3.2?mM of pyruvic acid, acrylic acid, propanoic acid, 2-oxo-butanoic acid, and 2-oxo-octanoic acid, respectively. Contrarily, the cresolase activity of MT was inhibited by all type of the above acids. Propanoic acid caused an uncompetitive mode of inhibition (K_i=0.14?mM), however, the pyruvic, acrylic, 2-oxo-butanoic and 2-oxo-octanoic acids showed a competitive manner of inhibition with the inhibition constants (K_i) of 0.36, 0.6, 3.6 and 4.5?mM, respectively. So, it seems that, there is a physical difference in the docking of mono- and o-diphenols to the tyrosinase active site. This difference could be an essential determinant for the course of the catalytic cycle. Monophenols are proposed to bind only the oxyform of the tyrosinase. It is likely that the binding of acids occurs through their carboxylate group with one copper ion of the binuclear site. Thus, they could completely block the cresolase reaction, by preventing monophenol binding to the enzyme. From an allosteric point of view, n-alkyl acids may be involved in activation of MT catecholase reactions.     

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.     

Kinetic study of sinephrine oxidation by mushroom tyrosinase

Mushroom tyrosinase catalyzes the oxidation of sinephrine showing a marked lag period during appearance of adrenochrome and simultaneously adrenaline accumulation in the reaction medium can be detected. The adrenaline accumulation follows a sigmoidal curve until a constant level of adrenaline is reached when the system is in the steady-state. These experimental results agree with a model of enzymatic catalysis that includes the chemical evolution of adrenoquinone and permit us to explain these phenomenon as well as the influence that enzyme and sinephrine concentration present on the lag period and the level of adrenaline accumulated in the steady-state.     

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.     

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     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

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.     

Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase

This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtained. This study with tyrosinase enzyme, which acts on L-dopa and catechol allowed us to determine the corresponding kinetic parameters, indicating that catechol is about 8-times more powerful as a suicide substrate than is L-dopa.     

Synthesis,molecular docking studies of coumarinyl-pyrazolinyl substituted thiazoles as non-competitive inhibitors of mushroom tyrosinase

A series of coumarinyl-pyrazolinyl substituted thiazoles derivatives were synthesized and their inhibitory effects on the DPPH and mushroom tyrosinase were evaluated. The results showed that all of the synthesized compounds exhibited significant mushroom tyrosinase inhibitory activities. In particular, 3-(5-(4-(benzyloxy)-3-methoxyphenyl)-1-(4-(4-bromophenyl)thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one (7j) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value 0.00458 ± 0.00022 μM compared with the IC₅₀ value of kojic acid is 16.84 ± 0.052 μM. The inhibition mechanism analyzed by Lineweaver–Burk plots revealed that the type of inhibition of compound 7j on tyrosinase was noncompetitive. The docking study against tyrosinase enzyme was also performed to determine the binding affinity of the compounds. The compound 7a showed the highest binding affinity (−10.20 kcal/mol) with active binding site of tyrosinase. The initial structure activity relationships (SARs) analysis suggested that further development of such compounds might be of interest. The statistics of our results endorses that compound 7j may serve as a structural template for the design and development of novel tyrosinase inhibitors.     

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.     

Kinetic study on the suicide inactivation of tyrosinase induced by catechol

Tyrosinase has a suicide inactivation reaction when it acts on omicron-diphenols. In the present paper, this reaction has been studied using a transient phase approach. Explicit equations of product vs. time have been developed for the multisubstrate mechanism of tyrosinase, and the kinetic parameters which characterize the enzyme acting on the suicide substrate catechol have been determined. The effect of pH has also been considered.     

Cyclopropylamine inactivation of cytochromes P450: role of metabolic intermediate complexes

The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5' (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to alpha-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1.