Mechanistic Explanation of Activation and Inhibition of Crucial Enzymes Involved in Melanin Production

ABSTRACT: Tyrosinase and tyrosinase-related protein-2 (Tyrp2) play a pivotal role in melanin production within melanocytes. Both share a high degree of structural and sequence similarity, with a Root-Mean-Square Deviation (RMSD) of 0.75 Å and 62% sequence similarity, including 43% sequence identity. Despite these similarities, differences in the allosteric sites, particularly the residues that constitute these sites, contribute to distinct regulatory mechanisms. This study elucidates the contrasting allosteric modulation of tyrosinase and Tyrp2 by two flavonoids: (a) luteolin and (b) apigenin, respectively. Luteolin demonstrated its allosteric inhibitory property against tyrosinase by disturbing the spatial arrangement of histidine residues critical for copper coordination, essential for enzymatic activity. Conversely, apigenin serves as an allosteric activator for Tyrp2, enhancing structural stability and maintaining the active site conformation. Despite the structural resemblance between luteolin and apigenin, differentiated by a single hydroxyl group, their impact on melanin synthesis diverges — luteolin inhibits, whereas apigenin stimulates the process. Both showed high binding affinity at the allosteric site of tyrosinase and Tyrp2. Luteolin significantly alters the spatial arrangement of key histidine residues (HIS180, HIS202 and HIS211), forming coordination bonds in tyrosinase, affecting copper coordination and coordination with the peroxide (O₂2⁻) ion. However, apigenin did not remain bound to tyrosinase for an extended period, ruling out its role as an allosteric modulator. In contrast, apigenin stayed at the allosteric site of the Tyrp2 protein and showed a positive allosteric regulator role. The findings offer a mechanistic view of melanin synthesis regulation, demonstrating the potential of subtle structural variations in modulators to selectively influence enzyme activity in related biosynthetic pathways. This underscores the intricate control within melanogenic systems and the possibility of targeting these pathways for therapeutic and cosmetic applications.