*4.2. DFT Calculation: Tyr, Trp, Met, and Cys Are Active Sites*

Theoretically, quantum chemical computations can gain prediction of behaviors of organic compounds, such as their structural features and chemical reactivity, and therefore, help to analyze the relationship between the biological potencies and the type of compounds [38]. The distributions of HOMO correspond to the active sites of the peptides able to scavenge free radicals [26]. By our DFT calculations, HOMOs of the tested peptides are located at their active amino acids, that is, Cys, Trp, Met, or Tyr (see Figure 2). The HOMOs of some peptides including EAAY, PMRGGGYHY, PMRGGYHY, PMRGYHY, PMRYHY, and YHY have been reported to be concentrated on the phenolic hydroxyl structure in Tyr [41]. The peptides PVETVR, QEPLLR, RDPEER, and LDDDGRL have the HOMOs of guanidyl in Arg, and the active sites of peptides KELEEK, DAAGRLQE, and GFAGDDAPRA are located at Lys-Glu, Gly, and Asp [24,39]. Clearly, data from HOMOs addressed that the residues Cys, Trp, Met, or Tyr are key components responsible for the antioxidant activity of the tested peptides in our study.

Generally, a high EHOMO or a low E-gap value means flexible chemical reactivity and could be used to predict the antioxidant activity of each peptide [24]. As predicted in our study, seven synthetic dipeptides, having a higher EHOMO and a lower E-gap value, showed a good ability to inhibit the oxidation of linoleic acid. It is found the seven antioxidant dipeptides possess the active residues Tyr, Trp, or Met. The presence of Tyr, Trp, and Met significantly enhanced the antioxidant activity of these dipeptides compared to other tested peptides (see Table 3). In a similar study, Wang et al. used EHOMO and E-gap to predicate the antioxidant activity of five peptides with only one exception [39]. Experiments conducted by Wu et al. also indicated that EHOMO and E-gap were feasible to describe the antioxidant behaviors of a set of man-made peptides, which were designed from the parent peptide "PMRGGGGYHY" [41]. Consistent with other studies reported [44], the presence of active residues Tyr, Trp, or Met as well as high EHOMO and low E-gap should be the characteristics of a peptide responsible for inhibiting the oxidation of linoleic acid. Amino acids, Tyr and Trp, act as active sites were also confirmed by Molecular docking. Wang et al. found that Trp1 and Tyr4 in peptide WLSYPMNPATGH could form hydrogen bonds with DPPH, which means responsible of Trp and Tyr in scavenging DPPH free radical. These two emerging approaches are helpful in analyzing antioxidative products, meanwhile, DFT calculation is a useful tool in screening antioxidant peptides [45].
