## 3 Results and Discussion
In the case of chemical compounds (L-cysteine and ascorbic acid), the received results were similar (regarding the type of inhibition). Only in the case of kojic acid was a different type of inhibition observed, dependent on the substrate used. In most scientific publications, synthetic substrates (pure chemical compounds such as catechol, pyrocatechol, or 4-methylcatechol) are used to conduct reactions with PPO (determining its activity, determining the IC\({}_{50}\) values, identifying the type of inhibition, and determining the kinetic constants of the process). In our study, we aimed to verify whether similar results would be obtained using an endogenous substrate obtained from plant material. IC\({}_{50}\) values were determined for selected inhibitors in the presence of the endogenous substrate. Interestingly, in the case of natural extracts, no statistically significant differences were observed in the reaction with exogenous (pyrocatechol) and endogenous substrates. However, it is worth noting that the IC\({}_{50}\) values determined for chemical compounds (L-cysteine, kojic acid, and ascorbic acid) in reaction with the endogenous substrate were significantly higher than in reaction with pyrocatechol as a substrate. Additionally, for the three most effective inhibitors (with the lowest IC\({}_{50}\) values: lovage, oregano, and orange peel), the inhibition model was determined in reaction with the endogenous substrate, and, next, the values of the kinetic constants were calculated. The obtained results are not identical to those obtained in the reaction with pyrocatechol. Some examples of Lineweaver-Burk plots for PPO inhibition (reaction carried out with endogenous substrate and pyrocatechol) were presented in Figure 2. In turn, the inhibition model and kinetic parameters of the process using an endogenous substrate were presented in Table 3.  
The results suggest that the use of endogenous substrates may be more appropriate for studying enzyme kinetics, especially in practical applications where reactions occur under natural conditions. The use of synthetic substrates may introduce certain distortions in the analysis of enzymatic kinetics because the reaction conditions may differ from those occurring in real biological systems. Therefore, studies demonstrating the effectiveness of endogenous substrates may suggest the need to consider alternative research methods that better reflect the natural environment of enzymes. Results regarding enzyme kinetics can also vary when different chemical compounds are used as substrates. [PERSON] et al. [32] examined the effect of natural anti-browning agents on PPO from ginger (_Zingiber officinal Roscoe_) and they report that the mechanism of honey in inhibiting PPO varies depending on the variety of substrates used. The V\({}_{\text{max}}\) value was unchanged and Km value was increased for PPO with honey using 4-methylcatechol. It can be concluded that honey showed competitive inhibition; however, honey exhibited non-competitive inhibition towards ginger PPO when pyrocatechol was used as the substrate, whereby the K\({}_{\text{m}}\) value was unchanged. This is another argument supporting the use of endogenous substrates for conducting reactions with PPO. Without more detailed research, including an analysis of the plant's phenolic profile, it is difficult to determine which phenolic compounds are utilized as substrates by enzyme. The effect of the selected metal ions at different concentrations varying from 20 to 5 mM (concentration in the reaction mixture: 2 mM to 0.5 mM) on iceberg lettuce PPO activity using pyrocatechol as a substrate was presented in Table 4. Unfortunately, the IC50 value for metal ions could not be determined, because they act in a dose-independent manner, whereas it was observed that, in the smallest studies concentrations (0.5 mM), MgCl\({}_{2}\) and FeCl\({}_{2}\) inhibit the activity of PPO by almost 30%, and KCl and NaCl by 18% and 17%, accordingly.