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 hinokitiol on tyrosinase activity and melanin biosynthesis and its antimicrobial activities

The inhibitory effects of hinokitiol, a constituent of the woody oils isolated from Cupressaceae heartwood, on mushroom tyrosinase and melanin formation in B16 melanoma cells as well as its antimicrobial activity were investigated. Our results showed that hinokitiol could strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition was reversible. The IC₅₀ values were estimated as 9.67?μM for monophenolase activity and 0.21?μM for diphenolase activity. The lag time of the monophenolase activity was not obviously lengthened by the compound. Kinetic analyses showed that the inhibition mechanism of hinokitiol was a mixed-type inhibition of the diphenolase activity. Hinokitiol effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells with significant cytotoxicity. Furthermore, it was found that hinokitiol could inhibit the proliferation of Salmonella enteritidis, Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, Klebsiella pneumoniae, and Ralstonia solanacearum to different extents. This research may widen the use of hinokitiol in the fields of food preservation, depigmentation, and insecticide use.     

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 4-Halobenzoic Acids on the Diphenolase and Monophenolase Activity of Mushroom Tyrosinase

The effects of 4-halobenzoic acids (4-fluorobenzoic acid, 4-chlorobenzoic acid, and 4-bromobenzoic acid) on the activity of mushroom tyrosinase have been studied. The results show that 4-halobenzoic acids can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme, and the inhibition displays a reversible course. The IC50 values were estimated as 0.26, 0.20, and 0.18 mM for diphenolase activity and as 1.03, 0.75, and 0.60 mM for monophenolase activity, respectively. Kinetic analyses show that the inhibition mechanism of all three 4-halobenzoic acids is noncompetitive inhibition to the diphenolase activity, and the inhibition constants (K1) were determined to be 0.25, 0.20, and 0.17 mM, respectively. The lag time of the monophenolase was obviously lengthened by these three 4-halobenzoic acids. When the concentration of inhibitors reached 1.4 mM, the lag time was lengthened from 30 s to 120, 125, and 150 s, respectively.     

Inhibitory effect of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on mushroom (Agaricus bisporus) tyrosinase

Mushroom tyrosinase (EC 1.14.18.1), a copper containing oxidase, catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the current study, the effects of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on the monophenolase and diphenolase activities of mushroom tyrosinase have been studied. The results show that 4-cyanobenzaldehyde and 4-cyanobenzoic acid can inhibit both the monophenolase activity and the 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. 1.0 mM 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lengthen the lag phase from 78 s to 134 and 115 s, respectively. Both 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lead to reversible inhibition of the enzyme. The IC50 values of 4-cyanobenzaldehyde and 4-cyanobenzoic acid were estimated as 0.62 and 2.45 mM for monophenolase and as 0.72 and 1.40 mM for diphenolase, respectively. A kinetic analysis shows that 4-cyanobenzaldehyde and 4-cyanobenzoic acid are mixed-type inhibitors for the diphenolase. The apparent inhibition constants for 4-cyanobenzaldehyde and 4-cyanobenzoic acid binding with both the free enzyme and the enzyme-substrate complex have been determined and compared.     

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 4-vinylbenzaldehyde and 4-vinylbenzoic acid 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 which form brown or black pigments. Here, the inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase have been investigated. The results showed that both 4-vinylbenzaldehyde and 4-vinylbenzoic acid could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, 4-vinylbenzoic acid could lengthen the lag time, but 4-vinylbenzaldehyde could not. Both 4-vinylbenzaldehyde and 4-vinylbenzoic acid decreased the steady-state activity, and the IC₅₀ values were estimated as 93?μM and 3.0?mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of 4-vinylbenzaldehyde was stronger than that of 4-vinylbenzoic acid, and the IC₅₀ values were estimated as 23?μM and 0.33?mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were mixed-II type; their inhibition constants were also determined and compared.     

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.     

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC₅₀ = 6.97 μM, monophenolase activity; IC₅₀ = 36.3 μM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.     

Inhibitory Effects of Hexylresorcinol and Dodecylresorcinol on Mushroom (Agaricus bisporus) Tyrosinase

The effects of hexylresorcinol and dodecylresorcinol on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that hexylresorcinol and dodecylresorcinol can inhibit both monophenolase and diphenolase activity of the enzyme. The lag period of the enzyme was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. Two microM of hexylresorcinol and dodecylresorcinol can lengthen the lag period from 98 s to 260 and 275 s, respectively. Both hexylresorcinol and dodecylresorcinol can lead to reversible inhibition of the enzyme. The IC50 values of hexylresorcinol and dodecylresorcinol were estimated as 1.24 and 1.15 microM for monophenolase and as 0.85 and 0.80 microM for diphenolase, respectively. A kinetic analysis shows that hexylresorcinol and dodecylresorcinol are competitive inhibitors. The apparent inhibition constant for hexylresorcinol and dodecylresorcinol binding with free enzyme has been determined to be 0.443 and 0.405 microM for diphenolase, respectively.     

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 μM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 μM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.     

Dual Effects of Alpha-Arbutin on Monophenolase and Diphenolase Activities of Mushroom Tyrosinase

The effects of α-arbutin on the monophenolase and diphenolase activities of mushroom tyrosinase were investigated. The results showed that α-arbutin inhibited monophenolase activity but it activated diphenolase activity. For monophenolase activity, IC₅₀ value was 4.5 mmol·L⁻¹ and 4.18 mmol·L⁻¹ of α-arbutin could extend the lag time from 40.5 s to 167.3 s. Alpha- arbutin is proposed to be regarded as a triphenolic substrate by the enzyme during catalyzation, leading to the suicide inactivation of the active site of tyrosinase. For diphenolase activity, α-arbutin acted as an activator and its activation mechanism was mixed type activation. To reveal such activation, it should be mainly refered to the conformational changes in tyrosinase caused by the interaction of α-arbutin with residues located at the entrance to the active site, and the decrease of the effect of suicide inactivation.     

Comprehensive kinetic and structural studies of different flavonoids inhibiting diphenolase activity of mushroom tyrosinase

The effect of 4 flavonoids on the diphenolase activity of mushroom tyrosinase was studied using spectroscopic approach. Analysis of kinetic data demonstrated that flavonoids cause a reversible inhibition of the enzyme activity. Further study showed that gallic acid acted as noncompetitive inhibitor, whereas chrysin, naringin and quercetin inhibited the diphenolase activity of mushroom tyrosinase in a competitive fashion. Comparison of the inhibition constants revealed that the strength with which the inhibitors acted on the enzyme activity was ranking as follows: chrysin (K_i 7.90 mM) < quercetin (K_i 7.44 mM) < naringin (K_i 3.04 mM) < gallic acid (K_i 1.5 mM). These data, therefore, suggest that gallic acid is the most potent inhibitor of the enzyme compared to the other flavonoids used.     

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 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.     

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.     

Inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid 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 which form brown or black pigments. Here, the inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase have been investigated. The results showed that both 4-vinylbenzaldehyde and 4-vinylbenzoic acid could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, 4-vinylbenzoic acid could lengthen the lag time, but 4-vinylbenzaldehyde could not. Both 4-vinylbenzaldehyde and 4-vinylbenzoic acid decreased the steady-state activity, and the IC50 values were estimated as 93 microM and 3.0 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of 4-vinylbenzaldehyde was stronger than that of 4-vinylbenzoic acid, and the IC50 values were estimated as 23 microM and 0.33 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were mixed-II type; their inhibition constants were also determined and compared.     

In Vitro Effect of Ozagrel on Mushroom Tyrosinase

This investigation, in vitro, shows that ozagrel, an antithrombotic drug, inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when l-tyrosine and l-DOPA were assayed spectrophotometrically, respectively. The IC₅₀ values, for monophenolase and diphenolase activities, were 1.35 and 3.45 mM, respectively. Ozagrel was estimated to be a reversible mixed-type inhibitor of diphenolase activity with the constants (K _S1, K _S2, K _i1, and K _i2) determined to be 2.21, 3.89, 0.454, and 0.799 mM, repectively. Increasing ozagrel concentrations provoked longer lag periods as well as a concomitant decrease in the monophenolase activity. Inhibition experiment demonstrated that ozagrel bound the enzyme at a site distincted from the substrate active site, but it bound to either E (Enzyme) or ES (Enzyme-Substrate) complex.     

Interaction of sesamol (3,4-methylenedioxyphenol) with tyrosinase and its effect on melanin synthesis

Sesamin, sesamolin (lignans) and sesamol - from sesame seed (Sesamum indicum L.) - are known for their health promoting properties. We examined the inhibition effect of sesamol, a phenolic degradation product of sesamolin, on the key enzyme in melanin synthesis, viz. tyrosinase, in vitro. Sesamol inhibits both diphenolase and monophenolase activities with midpoint concentrations of 1.9 μM and 3.2 μM, respectively. It is a competitive inhibitor of diphenolase activity with a K_i of 0.57 μM and a non-competitive inhibitor of monophenolase activity with a K_i of 1.4 μM. Sesamol inhibits melanin synthesis in mouse melanoma B16F10 cells in a concentration dependant manner with 63% decrease in cells exposed to 100 μg/mL sesamol. Apoptosis is induced by sesamol, limiting proliferation. This study of the chemistry and biology of lignans, in relation to the mode of action of bioactive components, may open the door for drug applications targeting enzymes.     

Inhibitory effects of phloridzin dihydrate on the activity of mushroom (Agaricus bisporus) tyrosinase

The inhibitory effects of phloridzin dihydrate on the activity of mushroom tyrosinase have been studied. The results show that phloridzin can inhibit the diphenolase activity of the enzyme and the inhibition displays to be reversible. The IC(50) value was estimated as 110microM. The kinetic analysis showed that the inhibition of phloridzin on the diphenolase activity of the enzyme is of competitive type, and the inhibition constant (K(I)) was determined to be 64.3microM. The inhibitory effects of the different concentrations of phloridzin on the monophenolase activity were also studied. There were almost no changes in the lag period and the steady-state rate, while the plateaus in the inhibitory curve lowered with increasing the concentration of phloridzin when using tyrosine as a substrate.