Previous studies showed a sequence encoding an auxiliary protein (PlaS) downstream of the phospholipase A1 (PlaA1) gene of Serratia marcescens. There is an interaction between PlaA1 and PlaS, which may be closely related to the high enzymatic activity property of phospholipase A1. In order to further investigate the interaction mechanism, it is necessary to explore binding sites of the interaction between PlaA1 and PlaS and the regulatory mechanism for enzymatic properties by molecular docking and site-directed mutagenesis. The results showed that the active center site of PlaA1 was encapsulated internally, and a “catalytic pocket” was formed externally by Leu197-Ser249. The docking process of PlaA1 and PlaS involved 29 and 30 amino acids, respectively, of which Phe186 and Lys238 of PlaA1 are involved in forming π-bonds and multiple hydrogen bonds. Therefore, Phe186 and Lys238 were site-directed mutated to Ala to obtain the mutant enzymes PlaA1 F186A and PlaA1 K238A, respectively. The results showed that the mutant enzymes showed no significant changes in optimum temperature and pH but poor stability. The kinetic parameters indicated that the affinity between PlaA1 and substrates was weakened, and the catalytic efficiency was reduced after mutation. Therefore, it demonstrated that Phe186 and Lys238 of PlaA1 provided non-covalent bonds conducive to the enzymatic activity and stability in the interaction between PlaA1 and PlaS, which would provide some theoretical basis for further rational design and modification of phospholipase A1 subsequently.