Oxidation of tyrosine residues in proteins by tyrosinase. Formation of protein-bonded 3,4-dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine.

A simple and rapid method was developed for the determination of 3,4-dihydroxyphenylalanine (dopa) and 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-cysteinyldopa) in proteins with the use of high-pressure liquid chromatography. With this method, it is demonstrated that mushroom tyrosinase can catalyse hydroxylation of tyrosine residues in proteins to dopa and subsequent oxidation to dopaquinone residues. The dopaquinone residues in proteins combine with cysteine residues to form 5-S-cysteinyldopa in bovine serum albumin and yeast alcohol dehydrogenase, whereas dopa is the major product in bovine insulin, which lacks cysteine residues.     

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.     

Hydroxylation of tyrosine by plant peroxidase and mushroom tyrosinase, with and without hydrazine, to retard the oxidation of dopa.

Tyrosine was oxidized to dopa by horseradish peroxidase and mushroom tyrosinase. The first step of hydroxylation of tyrosine in the synthesis of melanin was demonstrated by isolation of dopa from the reaction mixture using hydrazine as a selective retardant.     

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.     

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.     

A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas

The unstable quinones of 3,4-dihydroxyphenylalanine (dopa) and the most abundant cysteinyldopa isomers (2S-, 5S- and 2,5S,S'-) have been generated rapidly via disproportionation of their respective semiquinones prepared pulse radiolytically by one-electron oxidation of the corresponding dopas with azide radicals. Dopaquinone decays via a base-catalysed unimolecular cyclisation yielding leucodopachrome which, under the present conditions, is immediately oxidised by remaining dopaquinone to form dopachrome and dopa back again. Addition of cysteine increased the rate of dopaquinone decay and precluded dopachrome formation. By contrast, the cysteinyldopa quinones decayed via an acid-catalysed unimolecular cyclisation involving the cysteine side chain to form a cyclic quinone-imine observed directly for the first time. These quinone-imine intermediates subsequently rearranged to more stable phenolic benzothiazine isomers. The addition of cysteine had little effect on cysteinyldopa quinone decay and did not prevent quinone-imine formation. The absorption spectra, extinction coefficients and rate constants for formation and decay of these various transient species involved in melanisation are reported.     

Pseudoperoxidase activity of mushroom tyrosinase

Under anaerobic conditions, ethyl hydroperoxide functions as a two-electron acceptor in the tyrosinase-catalyzed oxidation of 4-tert-butylcatechol to 4-tert-butyl-o-benzoquinone, apparently by the following mechanism:

$\text{T?[Cu(II)]}{}_2\text{+ TBC = T?[Cu(I)]}{}_2\text{+ TB?}\text{o?}\text{BQ + 2H}{}^{\mathrm{+}}$

$\text{T?[Cu(I)]}{}_2\text{+ EtOOH + 2H}{}^{\mathrm{+}}\text{= T?[Cu(II)]}{}_2\text{+ EtOH +H}{}^2\text{O}$

This is a direct demonstration of the pseudoperoxidase activity of tyrosinase. Ethyl hydroperoxide failed to oxidize either oxy- or deoxyhemocyanin.     

Synthesis of tyrosine and 3,4-dihydroxyphenylalanine by bacteria Citrobacter freundii

Among facultative-anaerobic bacteria utilizing formic acid, a large number of strains having tyrosine phenol lyase were found. The enzyme can catalyze synthesis of tyrosine and 3,4-dihydroxy phenyl alanine (DOPA) from pyruvate, ammonium and, accordingly, phenol and pyrocatechol. These strains were identified as Citrobacter freundii. Cell suspensions of the most active strains synthesized up to 75 g/l tyrosine for 12 hr, up to 86 g/l tyrosine for 24 hr, and up to 29 g/l DOPA for 42 hr. A medium containing yeast autolysate grown on hydrocarbons can be recommended to produce cells having a high tyrosine phenol lyase activity.     

Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.     

3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system

1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.     

Reactions of DOPA (3,4-dihydroxyphenylalanine) decarboxylase with DOPA.

The study of DOPA (3,4-dihydroxyphenylalanine) decarboxylase by steady-state methods is difficult because multiple reactions occur. The reaction with DOPA was studied at enzyme concentrations between 20 and 50 micrometer by direct observation of the bound coenzyme by using stopped-flow and conventional spectrophotometry. Four processes were observed on different time scales and three of these were attributed to stages in the decarboxylation. The fourth was attributed to an accompanying transamination that renders the enzyme inactive. It was clear that much, if not all, of the 330 nm-absorbing coenzyme present in the free enzyme plays an active part in the decarboxylation, since it is converted into 420 nm-absorbing material in the first observable step. An intermediate absorbing maximally at 390 nm is formed in a slower step. Rate and equilibrium constants have been determined and the ratio of decarboxylation to transamination was estimated to be 1200:1.     

Synthesis and biological evaluation of helicid analogues as mushroom tyrosinase inhibitors

A series of helicid analogues were synthesized and evaluated as tyrosinase inhibitors. The results demonstrated that some compounds had more potent inhibitory activities than arbutin (IC(50) 7.3 mM). In particular, compound 1c bearing 4,6-O-benzylidene substituent on the sugar moiety was found to be the most potent inhibitor with IC(50) value of 0.052 mM. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that helicid analogues were competitive inhibitors. The Circular dichroism spectra indicated that those compounds induced conformational changes of mushroom tyrosinase upon binding.     

Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase

Free tyrosine and tyrosine residues in various peptides and proteins are converted into dopa and dopa residues by tyrosinase (monophenol,L-dopa:oxygen oxidoreductase, EC 1.14.18.1) in the presence of reductants. The efficiency of the tyrosine-to-dopa conversion was examined under varied conditions, such as the substrate-to-tyrosine ratio, concentrations of reductant and oxygen in the reaction solution, pH, temperature and reaction time. The highest dopa yields were achieved with the following optimal conditions for hydroxylation: 0.1 M phosphate buffer at pH 7, 25 mM ascorbic acid, 1 mM tyrosine, 50 micrograms/ml tyrosinase and 20 degrees C. Using these conditions, up to 70% of free tyrosine was converted into dopa, and tyrosine residues in several synthetic peptides were also hydroxylated to dopa residues at ratios as high as free tyrosine. The preparation of hydroxylated analogues of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys), in particular, may contribute to a better understanding of adhesion in the dopa-containing mussel glue protein.     

Production of a chiral alcohol, 1-(3,4-dihydroxyphenyl) ethanol, by mushroom tyrosinase

1-(3,4-Dihydroxyphenyl) ethanol was produced biocatalytically for the first time using mushroom tyrosinase. 4-Ethylphenol at 1 mM was consumed over 12 min giving 0.23 mM 4-ethylcatechol and 0.36 mM (R/S)-1-(3,4-dihydroxyphenyl) ethanol (ee 0.5 %). Mushroom tyrosinase consumed 4-ethylphenol at 6.7 μmol min^?1 mg protein^?1 while the rates of formation of 4-ethylcatechol and 1-(3,4-dihydroxyphenyl) ethanol were 1.1 and 1.9 μmol min^?1 mg protein^?1. Addition of the ascorbic acid, as a reducing agent to biotransformation reactions, increased 4-ethylcatechol formation by 340 %. However, accumulation of 1-(3,4-dihydroxyphenyl) ethanol was not observed in the presence of ascorbic acid. While the 1-(3,4-dihydroxyphenyl) ethanol was racemic, it is the first chiral product produced by tyrosinase starting from a non-chiral substrate.     

Synthesis of tyrosine and 3,4-dihydroxyphenylalanine by Citrobacter freundii bacteria from lactic acid

A possibility of using lactic acid as a precursor for the synthesis of L-tyrosine and L-3,4-dihydroxyphenylalanine (DOPA) by Citrobacter freundii 62 and 63 was established. The synthesis of tyrosine from lactic acid occurs at a phenol concentration of less than 0.6%. The conditions were found which enable C. freundii 62 and 63 to synthesize from lactic acid tyrosine and DOPA with the yield of 35-38 g/l and 32-33 g/l, respectively.     

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.     

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.