Human TYR is a glycoprotein (13% carbohydrate) predominantly located in the melanosome membrane of melanocytes [23,24]. The presence of the transmembrane domain and glycans renders it difficult to isolate homogeneous TYR from melanocytes, which impedes crystallographic studies. Fortunately, the most characteristic TYR can be acquired from Streptomyces glausescens, the fungi Neurospore crassa, or Agaricus bisporus [7,25,26]. To compare the conservation of catalytic cavity, the crystal structures of TYR from bacteria (Streptomyces castaneoglobisporus [25] and Bacillus megaterium [27]), fungi Agaricus bisporus [28], and walnut leaves [29] were retrieved from Protein Date Bank. A salient feature of TYR from various sources (Figure 3) is the presence of strictly conserved binuclear copper atoms at the active site, each of copper atoms is coordinated with three conserved histidines, respectively [3]. Moreover, the normal redox state of copper atoms is exceedingly significant for enzyme activity. Due to the relatively high similarity and homology with mammalian TYR, mushroom TYR from Agaricus bisporus acts as a model for enzyme kinetics and inhibitor screening [28,30,31]. Although extensive studies are devoted to TYR, its catalytic mechanism remains controversial. According to the presence/absence of oxygen and the oxidation state of copper ions [Cu (Ⅱ)/Cu (Ⅰ)], three enzymatic forms (E_oxy, E_met, and E_deoxy) participate in the catalytic cycle (Figure 4) [3,32]. The resting form of TYR is found to be a mixture of 85% met and 15% oxy forms, while only the latter could act on the monophenol [7,33,34]. During the monophenolase cycle, to form E_oxy-monophenol complex (E_oxyM), the oxygen atom on the deprotonated monophenol is coordinated with the coppers of E_oxy. Then, the phenol is o-hydroxylated to generate E_met-diphenol (D) complex (E_metD) [26,35]. Reducing agents could well draw the E_met into the E_deoxy, with concomitant oxidation to the corresponding o-quinone [36]. Since the deoxy form is the only one capable of reacting with oxygen to regenerate E_oxy and continue the catalytic action, monophenolase activity usually manifests as a characteristic lag time until a sufficient amount of catechol helps E_met to become E_deoxy [36]. Remarkably, this period depends on several factors, including enzyme concentration, monophenol concentration and the presence of reducing agents, especially o-diphenol derivatives (such as L-DOPA) that could shorten and even abolish the lag time [30,33,37]. In the diphenolase cycle, E_oxy continues to bind o-diphenol to originate the E_oxyD complex, while both E_oxy and E_met are capable of oxidizing the diphenol to the o-quinone. After this, E_met is regenerated to complete the catalytic cycle continuously [30,34].

Based on the broad substrate spectrum, in principle, any simple monophenol or corresponding catechol appears to be its substrate [38]. Besides, TYR also oxidizes various aromatic amines, o-aminophenols, and aromatic o-diamines (Figure 5), despite the reaction rates being orders of magnitude smaller than the corresponding phenols or catechol [39,40]. In terms of phenols, mammalian TYR tends to be relatively specific for its physiological substrate (L-tyrosine and L-DOPA) and has a higher affinity for the L-isomers [41]. A prevalent characteristic in monophenol substrates is without substituents in the ortho-position of the phenolic hydroxyl group. Understandably, large side-chain substituents increase the difficulty of substrate interaction with the key catalytic residues; this is unpropitious for the recognition and catalytic process between the enzyme and ligand [36]. A kinetic study [42] quantitatively discussed the effects of substituents in the 1-position of the aromatic ring on the rate of hydroxylation catalyzed by TYR. The results revealed that monophenols with a high electron donor tend to be oxidized faster [42]. In sharp contrast, the oxidation rate of catechol is positively correlated with the electron-withdrawing capacity of the para-substituents [36]. As such, the steric hindrance, stereochemical characteristics, and electronic effects of substituents have a distinct influence on the rate of TYR-mediated catalysis.