Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.
Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.
Olivares
Concepción
C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, 30100 Murcia, Spain.
García-Borrón
José C
JC
Solano
Francisco
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008546:Melanoma, Experimental; D008956:Models, Chemical; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D014162:Transfection
D008024:Ligands; D014442:Monophenol Monooxygenase