Ferroxidase activity of mushroom tyrosinase

Mushroom tyrosinase catalyses the oxidation of Fe(II) to Fe(III). Both the newly-discovered ferroxidase and the well-characterized diphenol oxidase activities of tyrosinase exhibit inhibition by cyanide and both activities co-purify during two preparation steps. The characteristics of tyrosinase-catalysed Fe(II) oxidation are compared with those of other ferroxidases.     

Effect of captopril on mushroom tyrosinase activity in vitro

The study presented here demonstrates that the antihypertensive drug captopril ([2S]-N-[3-mercapto-2-methylpropionyl]-L-proline) is an irreversible non-competitive inhibitor and an irreversible competitive inhibitor of the monophenolase and diphenolase activities of mushroom tyrosinase when L-tyrosine and L-DOPA were assayed spectrophotometrically in vitro, respectively. Captopril was rendered unstable by tyrosinase catalysis because of the interaction between the enzymatic-generated product (o-quinone) and captopril to give rise to a colourless conjugate. Therefore, captopril was able to prevent melanin formation. The spectrophotometric recordings of the inhibition of tyrosinase by captopril were characterised by the presence of a lag period prior to the attainment of an inhibited steady state rate. The lag period corresponded to the time in which captopril was reacting with the enzymatically generated o-quinone. Increasing captopril concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady state rate were dependent of captopril, substrate and tyrosinase concentrations. The inhibition of both monophenolase and diphenolase activities of tyrosinase by captopril showed positive kinetic co-operativity which arose from the protection of both substrate and o-quinone against inhibition by captopril. Inhibition experiments carried out using a latent mushroom tyrosinase demonstrated that captopril only bound the enzyme at its active site. The presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition. This could be due to the formation of both copper-captopril complex and disulphide interchange reactions between captopril and cysteine rich domains at the active site of the enzyme.     

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.     

Successful resonance Raman study of cresolase activity of mushroom tyrosinase

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.     

Inhibitory effects of naphthols on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1), a copper-containing multifunctional oxidase, was known to be a key enzyme for biosynthesis in fungi, plants and animals. In this work, the inhibition properties α-naphthol and β-naphthol toward the activity of tyrosinase have been evaluated, and the effects of α-naphthol and β-naphthol on monophenolase and diphenolase activity of tyrosinase have been investigated. The results showed that both α-naphthol and β-naphthol could potently inhibit both monophenolase activity and diphenolase activity of mushroom tyrosinase, and that β-naphthol exhibited stronger inhibitory effect against tyrosinase than α-naphthol. For monophenolase activity, β-naphthol could not only lengthen the lag time but also decrease the steady-state activity, while α-naphthol just only decreased the steady-state activity. For diphenolase activity, both α-naphthol and β-naphthol displayed revisible inhibition. Kinetic analyses showed that both α-naphthol and β-naphthol were competetive inhibitors.     

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     

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.     

Quaternary structure of mushroom tyrosinase

The quaternary structure of $\text{Agaricus}$$\text{bispora}$ tyrosinase has been investigated by sodium dodecylsulfate-acrylamide gel electrophoresis. The enzyme was found to contain two types of polypeptide chains, referred to as Heavy, molecular weight 43,000 ± 1,000, and Light, molecular weight 13,400 ± 600. In aqueous solution the predominant form of tyrosinase m.w. 120,000, has the quaternary structure L₂H₂.     

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.     

Effect of tiron on monohydroxy and o-dihydroxyphenolase activity of mushroom tyrosinase

Tiron has a multiple effect on mushroom tyrosinase. At relatively low concentrations (up to 3.3 mM), Tiron extended the lag period of tyrosine hydroxylation appreciably, while at concentrations between 3.3 and 8.3 mM the lag period was shortened and approached that of the control. At concentrations above 10 mM, Tiron shortened the lag period of tyrosine hydroxylation compared with that of the control.Tiron, at relatively high concentrations (above 266 mM), inhibited the initial rate of dl-DOPA oxidation by mushroom tyrosinase and lowered the final level of dopachrome formed. Preincubation of mushroom tyrosinase with Tiron resulted in the inactivation of the enzyme, with 50 % inactivation of 650 μg enzyme occurring in the presence of 400 mM Tiron.     

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.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.     

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, the effects of Cefazolin and Cefodizime on the activity of mushroom tyrosniase have been studied. The results showed that the Cephalosporin antibacterial drugs (Cefazolin and Cefodizime) could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, Both Cefazolin and Cefodizime could lengthen the lag time and decrease the steady-state activities, and the IC₅₀ values were estimated as 7.0 mM and 0.13 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of Cefodizime was obviously stronger than that of Cefazolin, and the IC₅₀ values were estimated as 0.02 mM and 0.21 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were competitive and mixed-type, respectively. Their inhibition constants were also determined and compared. The research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors and also under the application field of Cephalosporins.     

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, the effects of Cefazolin and Cefodizime on the activity of mushroom tyrosniase have been studied. The results showed that the Cephalosporin antibacterial drugs (Cefazolin and Cefodizime) could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, Both Cefazolin and Cefodizime could lengthen the lag time and decrease the steady-state activities, and the IC(50) values were estimated as 7.0 mM and 0.13 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of Cefodizime was obviously stronger than that of Cefazolin, and the IC(50) values were estimated as 0.02 mM and 0.21 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were competitive and mixed-type, respectively. Their inhibition constants were also determined and compared. The research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors and also under the application field of Cephalosporins.     

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.     

In vitro translation of mushroom tyrosinase

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.     

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.     

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 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 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.