Tyrosinase (monopheno1, dihydroxyphenyl alanine: oxygen oxidoreductase) is a copper-containing monooxygenase enzyme catalyzing both the o-hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones. The enzyme occurs in different microorganisms, plants and animals and is mainly involved in the biosynthesis of melanins and other polyphenolic compounds 1.
Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionyl-threonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein 1.
The first hints of tyrosinase activity arose in the late 1800s when the French naturalists Bourquelot and Bertrand observed that the toadstool Russula nigricans bore a colourless substance which blackened upon exposure to air. A year after the discovery, Bertrand recognised the substance as tyrosine, and in 1904 – barely a year later – it was shown that extracts of a mammalian melanoma could convert tyrosine to melanin. Scientists were beginning to grasp the intricate process of melanogenesis, i.e. the synthesis of the natural pigment melanin. The British physiologist H.S.Raper carried out the most important studies on melanogenesis from 1920 to 1935, but it was only during the 1950s that some real sense was made out of the melanin biosynthetic pathway 2.
The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids. CB1 is the largest of the four cyanogen bromide fragments of Neurospora tyrosinase, containing 222 amino acid residues. As a consequence of its size, a large number of peptides (>140) had to be isolated to arrive at the primary structure. The cyanogen bromide fragment CB2 contains 82 of the 407 amino acid residues of Neurospora tyrosinase. It is devoid of histidine and homo- serine; hence, it is placed at the COOH-terminal end of the intact protein. The cyanogen bromide fragment CB3 is composed of 68 residues and is devoid of tryptophan. It contains an Asn-Gly bond (residues 18 and 19) whose presence was confirmed by the isolation of two fragments after hydroxylamine cleavage of peptide Thl. Finally, the amide assignments of the aspartic acid residues of CB3 were found to be in complete agreement with the known specificity of cleavage at Asp by dilute acetic acid. The cyanogen bromide fragment CB4 consists of 35 amino acid residues lacking lysine and tyrosine. It shows a high content of hydrophobic amino acids (438) including 3 out of a total of 12 tryptophans in Neurospora tyrosinase. This unusual amino acid composition could at least partially be responsible for the observed resistance of cleavage of the Arg 21- Leu 22 bond by trypsin 1.
Tyrosinase (192-200) (human, mouse) H-Ser-Glu-Ile-Trp-Arg-Asp-Ile-Asp-Phe-OH: Antigenic peptide presented by HLA-B44 and recognized on a human melanoma by autologous cytolytic T lymphocyte 3.
Natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of Asn to Asp 5.
Mode of Action
A possible alternative mechanism for synthesis of catecholamines involves tyrosinase, the enzyme responsible for the first step in melanin formation. In this step, tyrosinase catalyzes the oxidation of tyrosine to dopaquinone. L-dopa is produced from dopaquinone during the first of a series of redox reactions leading to the formation of melanin. L-dopa further drives the reaction by functioning as an alternative substrate for tyrosinase and as a cofactor that stimulates catalytic efficiency. In contrast to lack of TH, lack of functional tyrosinase is not lethal and is commonly encountered as oculocutaneous albinism 6.
Tyrosinase is a multifunctional enzyme that catalyzes several steps in melanin production, the first and crucial step involving the hydroxylation of tyrosine. Although it is well established that L-dopa is produced during melanin synthesis, it is unlikely that tyrosinase is directly responsible for production of DA from L-dopa. A more reasonable explanation, supported by the present neurochemical and immunohistochemical data, is that tyrosinase-dependent DA production requires the additional actions of L-AADC to convert L-dopa to DA. Alternatively, since tyrosine is also a substrate for L-AADC, it is possible that tyrosine may first be decarboxylated to tyramine, then converted to dopamine by tyrosinase 6.
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