Effect Of Histidine Modification On Tyrosinase Activity And Conformation

Tyrosinase(EC 1.14.18.1),a ubiquitously expressed enzyme in organisms,plays a pivotal role in the melanin biosynthesis.The enzyme possesses multi-catalytic functions in the melanosynthetic pathway.Typically,two copper(Cu) ions,either as cupric or cuprous,interact individually with three histidine residues at the active site of the enzyme and are directly involved in the various catalytic reactions via the oxy-, deoxy- and met-states.Due to its complex multi-catalytic functions,the mechanism of tyrosinase catalysis remains elusive and the enzyme kinetics vary distinctively with different sources of tyrosinases.Currently,no crystallographic structure of the tyrosinase has been elucidated.Therefore,probing more into the catalytic mechanism as well as understanding regulations at the active site becomes significant.Based on the presence of free histidine and cysteine residues in the primary gene sequence of mushroom tyrosinase(GenBank accession number:AJ223816),it is speculated that these residues are not involved in the covalent binding and may be involved in the cooperative binding possibility of ligand and additive chemicals.It is not yet clear what exact role(s) of these amino acid residues plays in the catalytic mechanism.A reasonable hypothesis is perhaps that these residues somehow involve in the catalytic mechanism when substrates such as L-tyrosine or L-DOPA access to the active site.In this regard,we tried to elucidate the role of free histidine residues at the active site of tyrosinase in this study.We found that BrAc chemical modification of histidine residues of tyrosinase conspicuously inactivated enzyme activity.For more insights,bromoacetate as a histidine modifier was applied to study inhibition kinetics and to observe structural changes by ligand binding.As a result,we not only observed the significant inhibition of tyrosinase via histidine modification, but also detected the structural changes.The binding of the Br ion directly induced the exposure of hydrophobic surfaces and let to complete inactivation of the tyrosinase. These observations imply that the active site of the tyrosinase may be flexible.Based on the inhibition kinetics and the computational modeling predication,we deduced the role of free histidine residues during catalysis and reasoned that these residues are related to the substrate accessibility.These findings,together with the flexible property of the active site of the tyrosinase would provide new insights for more effective design of future tyrosinase inhibitors.