## 6 Papain Use as an Exfoliant Agent in Cosmetic Applications
### Bromelain, Ficin, and Papain Mechanisms of Action  
Bromelain, ficin, and papain, all examined in this study, are classified as plant-derived, cysteine proteinases, and belong specifically to the peptidase family C1 (papain family). The latex of papayas has been understood since 1880, and includes a large family of peptidases which are homologous to papain [122; 123; 124]. The dyad catalytic residues of the C1 family are the amino acids cysteine (Cys, C\({}_{3}\)H\({}_{7}\)NO\({}_{2}\)S) and histidine (His, C\({}_{6}\)H\({}_{9}\)N\({}_{3}\)O\({}_{2}\)). The two residues located at the active site are glutamine (Gln), which is positioned before Cys, and asparagine (Asn), located after His. His, in fact, is a semi-essential amino acid, while Cys is a non-essential, sulfuric residue. Cys, especially, comprises a thiol group, and thus is a unique amino acid residue (R-SH; sulfur takes the place of the oxygen in the hydroxyl). Gln is claimed to assist in \"oxygenion holes\" creation, whereas Asn helps to position the imidazolium ring of His (Gln and Asn residues are not illustrated in Figure 6) [122; 123; 124].  
Therefore, regarding their mechanism of action, even though they follow the same catalytic strategy, they have slight differences according to their optimal function conditions, stability, and substrate specificity, as reported. Bromelain exhibits broad substrate specificity, favoring peptide bonds adjacent to lysine alanine, tyrosine, and glycine basic amino acid residues [42], while it can hydrolyze proteins including gelatin, casein, and fibrinogen [125; 126]. Stem bromelain, for instance, preferentially cleaves substrates with arginine at the P1 and P2 positions [127]. Ficin, on the contrary, has a higher affinity for hydrophobic residues (i.e., phenylalanine (Phe)) located at the P1 position [128]. Similarly, papain has a broad specificity, cleaving basic, hydrophobic, and often polar residues, while working well on bulky amino acids like Phe [129]. All enzymes share high proteolytic activity and peptide bond hydrolysis patterns due to their structural similarity, having the same active sites and target proteins, and similar biochemical skin effects, as they are highly effective for skin exfoliation and renewal [22; 32; 91].  
The peptide bond hydrolysis process of papain family enzymes is stepwise, consisting of three stages: (1) the nucleophilic attack by the sulfur (thiol) group of cysteines on the peptide bond's carbonyl carbon, (2) the formation of a tetrahedral intermediate which is stabilized by Gln, His, and Asn, and (3) peptide bond cleavage, resulting in the release of smaller peptides or amino acids [123]. More specifically, after a peptide manages to bind to the active site, His-159 deprotonates Cys-25, allowing it to attack the carbonyl carbon in order to, again, create a tetrahedral intermediate, stabilized by an oxyanion hole (Figure 6(1)). Non-covalent-based interactions, such as hydrogen or ionic bonds, van der Walls forces, or hydrophobic interactions, along with Gln-19, aid in stabilizing the complex at its high-energy state. His-159, by acting as an acid, then protonates the nitrogen of the peptide bond, allowing it to act as a leaving group. This action concludes in carbonyl reformation and the release of the C-terminal peptide (Figure 6(2)). Water can subsequently access the active site and attack the carbonyl carbon once again, which is deprotonated again by His-159 in order to form another tetrahedral intermediate, stabilized by the oxyanion hole (Figure 6(3)). Ultimately, carbonyl is reformed, and Cys-25's sulfur behaves as the leaving group, releasing the N-terminal peptide portion and regenerating the enzyme (Figure 6(4,5)). The enzyme, as a catalyst, returns to its original conformation, ready to mediate another reaction cycle with a new substrate molecule (Figure 6(6)). The general mechanism of bromelain, ficin, and papain cysteine proteases catalysis is presented in Figure 6[29; 122; 124; 130].