## 3 Results and Discussion

Table 2: Effect of selected chemical compound and natural plant extracts on the iceberg lettuce PPO activity. Figure 2: Mode of PPO inhibition by selected natural extracts (Lineweaver–Burk plots)—effect of substrate used: (**A**) PPO inhibition by oregano extract (endogenous substrate); (**B**) PPO inhibition by oregano extract (pyrocatech as a substrate; (**C**) PPO inhibition by lavage extract (endogenous substrate); and (**D**) PPO inhibition by lavage extract (pyrocatech as a substrate). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{5}{c}{**Pyrocatech as a Substrate**} \\ \hline
**Inhibitor** & **V\({}_{\text{max}}\)** & **K\({}_{\text{init}}\)** & **V\({}_{\text{max}}\)** & **K\({}_{\text{in}}\)** & \\
**(IC50 Value)** & **[mU]** & **[mM]** & **[mU]** & **[mM]** & **Mode of Inhibition** \\ \hline L-cysteine & 0.87 & 174 & 0.91 & 21.4 & competitive \\ \hline kojic acid & 0.38 & 20 & 0.61 & 12.5 & mixed \\ \hline ascorbic acid & 1.37 & 202 & 1.39 & 52.4 & competitive \\ \hline basil infusion & 1.71 & 8.72 & 2.08 & 8.16 & mixed \\ \hline lavage infusion & 2.03 & 27.8 & 2.05 & 11.11 & mixed \\ \hline oregano infusion & 1.77 & 8.28 & 1.74 & 5.42 & competitive \\ \hline wheat bran infusion & 2.39 & 3.35 & 2.45 & 3.17 & competitive \\ \hline lemon peel infusion & 2.21 & 15.96 & 2.16 & 12.77 & non-competitive \\ \hline orange peel infusion & 625 & 3.2 & 714 & 3.6 & uncompetitive \\ \hline marjoram infusion & 0.37 & 5.38 & 0.66 & 2.13 & mixed \\ \hline parsley infusion & 1.12 & 9.07 & 3.93 & 25.18 & uncompetitive \\ \hline \hline  & \multicolumn{5}{c}{endogenous substrate} \\ \hline  & U & mg GAE/mL & U & mg GAE/mL & \\ \hline L-cysteine & 0.104 & 17.24 & 0.067 & 4.5 & competitive \\ \hline kojic acid & 0.0067 & 1.85 & 0.067 & 4.5 & non-competitive \\ \hline ascorbic acid & 0.034 & 2.94 & 0.067 & 4.5 & competitive \\ \hline lovage infusion & 0.0046 & 0.57 & 0.0098 & 0.625 & non-competitive \\ \hline oregano infusion & 0.0059 & 0.46 & 0.0098 & 0.61 & uncompetitive \\ \hline orange peel infusion & 0.0083 & 0.59 & 0.0098 & 0.0625 & competitive \\ \hline IC50—the concentration of a substance at which 50\% inhibition of enzyme activity is achieved, —inhibitor, V\({}_{\text{max}}\)—maximal velocity of reaction, K\({}_{\text{in}}\)—Michealis constant, U—unit, GAE\(-\)—galic acid equivalents. & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Mode of inhibition and kinetic parameters of process using pyrocatech and endogenous substrate. The results from the Lineweaver-Burk plots indicated that the L-cysteine and ascorbic acid acted as competitive inhibitors of PPO with a K\({}_{\rm mi}\) of 174 mM and 202 mM, respectively. The competitive inhibitor resembles the substrate molecule in its structure. It binds to the free enzyme in the active centre, preventing the substrate from binding to the enzyme molecule. Increasing the substrate concentration partially eliminates the inhibitor's effect. In the case of non-competitive inhibition, the inhibitor binds in a place other than the active centre. Increasing the substrate concentration does not eliminate the inhibitor's effect. In turn, uncompetitive inhibitors bind to the resulting enzyme-substrate complex, leading to the formation of an inactive enzyme-substrate-inhibitor complex. Moreover, in this case, increasing the substrate concentration does not eliminate the inhibitor's effect. Mixed inhibition is a case of partially competitive and non-competitive inhibition. The inhibitor can bind to both the free enzyme and the ES complex. In this case, kojic acid was observed to be of the mixed inhibition type. These results are in agreement with [PERSON] and [PERSON] [29], who reported that both cysteine and ascorbic acid are competitive inhibitors of PPO in fresh lettuce (_Latuca sativa_). [PERSON], [PERSON], [PERSON], and [PERSON] [30] also report that ascorbic acid in concentrations <1.5% acted as a competitive inhibitor, while, in a higher concentration, it could reduce the formed quinone instantly to the origin substrate (catechol).