To further test the reliability of identified residue sites, we selected eight high-attention sites on enzymes and performed single-site mutation verification. We mutated two proximal sites on each enzyme to alanine and found that, in most cases, enzyme activity significantly decreased after the mutation, indicating that these residues play important roles in catalysis (Fig and). Furthermore, the OH1_348 site was only identified by PU-EPP, suggesting that PU-EPP could be considered a supplement to MD for the identification of potentially important residues. Subsequently, we mutated two top-ranked distal sites on each enzyme to alanine and found that the enzymes' activity also decreased significantly, indicating that PU-EPP can identify distal residues that play important roles during catalytic processes, although most of them are generally considered to have no significant effect on catalytic activity (Fig 5I and). The attention weights on each atom in the ZEA/OTA molecules. The contour lines around the atoms represent the attention weights: the more contour lines, the higher the attention weight. Atoms near the reaction sites, such as the oxygen atom in the lactone bond of ZEA and nitrogen atom in the amido bond of OTA, both ranked 2nd in all the 45 and 46 atoms in ZEA and OTA, indicating these atoms contribute more to the final prediction. (E, F) The simplified UpSet plots of randomly selected residues and residues identified by PU-EPP and MD simulations, respectively. We evaluated the consistency of predicted residues and found ~90% of residues identified by PU-EPP on ZH4 (n = 56 for attention-identified residues and random residues, and n = 161 for MD-identified residues) and ~40% on OH1 (n = 124 for attention-identified residues and random residues, n = 131 for MD-identified residues) are consistent with MD, and both are significantly higher than randomly selected residues. Error bars represent ± s.d. (G, H) The boxplot and distribution of the hit ratio of atoms near reaction centers. It shows the median (horizontal line), 25th and 75th percentiles (lower and upper boundaries, respectively). Whiskers extend to data points that lie within 1.5 interquartile ranges of the 25th and 75th quartiles; and observations that fall outside this range are not displayed. The hit ratio of PU-EPP is significantly higher than that of random selection.