Deletion mutational mapping of the catalytic activities of human tyrosinase

M.-J. Paik, H.-J. Jo, J.-W. Lee, K.-H. Kong. Chung-Ang Univerity, Chemistry / 156-756, Republic of Korea

Tyrosinase is a multifunctional copper-containing glycoprotein, which plays a key role in the rate-limiting steps of the melanin biosynthetic pathway. Tyrosinase is related to a variety of disease, albinism or pigmentation in skin cell. In this study, we have constructed the four deletion mutants of human tyrosinase as follows; (1) a mutant lacking the C-terminal transmembrane and cytoplasmic domains (hTyr-TM); (2) a mutant lacking the C-terminal 455-481 amino acids, transmembrane and cytoplasmic domains (hTyr-C); (3) a mutant lacking the N-terminal 1-92 amino acids and the C-terminal transmembrane and cytoplasmic domains (hTyr-N); (4) a mutant lacking the N-terminal 1-92 amino acids and the C-terminal 455-481 amino acids, transmembrane and cytoplasmic domains (hTyr-C&N). The constructed four deletion mutants were expressed in E. coli and the recombinant human tyrosinase deletion mutants were produced as a fusion protein with His6-tag sequence. The four deletion mutants exhibited tyrosinase activities towards the hydroxylation and oxidation reactions. The deletion mutants also exhibited similar substrate specificity, showing high activities towards catechin, catechol, L-DOPA, and pyrogallol. The K m values for L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) of the deletion mutants were 1.32-2.7 μM and 0.27-0.34 mM, respectively. The optimum pHs of the deletion mutants were 7.5-8.5. The optimum temperatures of the deletion mutants were 40-60℃. From these results, we suggest that the human tyrosinase deletion domain consisting of only 92-455 amino acids is active, and residues in this domain play an important role in the active site.