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.     

A tyrosine hydroxylase assay in microwells using coupled nonenzymatic decarboxylation of dopa.

A radiometric assay for tyrosine hydroxylase employing a coupled nonenzymatic decarboxylation of L-[14C]Dopa formed from L-[14C]tyrosine has been adapted for performance in a 96 microwell culture plate. The method uses an easily manufactured plate holder to compress blotting paper impregnated with methylbenzethonium hydroxide against the top rim of each well. This forms isolated, airtight compartments in which 14CO2 is evolved and quantitatively absorbed into the blotting paper. The method is sensitive enough to detect the production of less than 5 pmol of 14CO2. A major advantage of this system is that cells can be grown in tissue culture and subsequently assayed for tyrosine hydroxylase activity in the same well. The method is more facile than previously devised procedures, allowing for the simultaneous assay of up to 96 samples totally contained in a single, compact, portable unit.     

A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon <Emphasis Type="Italic">Candidatus Nitrosopumilus koreensis</Emphasis>

Objectives

To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine at elevated pH and high temperature.

Results

A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis. This protein contains key tyrosinase domains, such as copper-binding domains and an O₂-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l^?1 and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio.

Conclusions

The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

     

Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

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.     

Influencing the monophenolase/diphenolase activity ratio in tyrosinase

Tyrosinase is a type 3 copper enzyme with great potential for production of commercially valuable diphenols from monophenols. However, the use of tyrosinase is limited by its further oxidation of diphenols to quinones. We recently determined the structure of the Bacillus megaterium tyrosinase revealing a residue, V218, which we proposed to take part in positioning of substrates within the active site. In the structure of catechol oxidase from Ipomoea batatas, the lack of monophenolase activity was attributed to the presence of F261 near CuA. Consequently, we engineered two variants, V218F and V218G. V218F was expected to have a decreased monophenolase activity, due to the bulky residue extending into the active site. Surprisingly, both V218F and V218G exhibited a 9- and 4.4-fold higher monophenolase/diphenolase activity ratio, respectively. X-ray structures of variant V218F display a flexibility of the phenylalanine residue along with an adjacent histidine, which we propose to be the source of the change in activity ratio.     

A point mutation in the tyrosine hydroxylase gene associated with Segawa's syndrome

We have examined the molecular basis of Segawa's syndrome in six families with seven affected children. In one family two siblings with this disease carried a point mutation in exon 11 of the tyrosine hydroxylase gene, resulting in an amino acid exchange of Gln³⁸¹ to Lys³⁸¹. These results suggest that a change in tyrosine hydroxylase causes this form of Segawa's syndrome.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.     

Characterization of dopa betaine,tyrosine betaine and N-dimethyltyrosine from Lobaria laetevirens

DOPA betaine, tyrosine betaine and N-dimethyltyrosine were isolated from the lichen Lobaria laetevirens. Their structures were determined by ra     

Reversible conversion of tyrosine hydroxylase to an inactive form by antipain

Tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamines, was reversibly inactivated by incubation with antipain, which is known as a microbial protease inhibitor. The inactivation was a time-dependent reaction and it was prominent when the enzyme was assayed at a neutral pH but not when assayed at an acidic pH. The inactivated enzyme was markedly activated by cyclic AMP-dependent protein kinase. Other microbial protease inhibitors such as leupeptin, chymostatin, and pepstatin did not induce such as inactivation of the enzyme.     

A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (BH(4)) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V(max)/K(m)) and the coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi-stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 A for the ensemble of docked conformers. Docking of BH(4) analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).     

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus,hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of ³H₂O from L-4-³H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of ³H₂O from L-4-³H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank ³H₂O produced from L-4-³H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank ³H₂O produced from L-3,5-³H-tyrosine. The K_m values of tyrosine hydroxylase for phenylalanine determined by the production of ³H₂O from L-4-³H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of ³H₂O formed from L-4-³H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.     

Detection of abnormally high amygdalin content in food by an enzyme immunoassay

Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.     

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.