The increase in the resistance of mutant strains of to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k/K, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k, and the weakening of the binding affinity, i.e., an increase in the substrate constant, K. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2 variant exhibits a higher k/K value, whereas for PBP2 and PBP2 variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k/K changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the β-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k/K value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the β-β loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k/K value. Thus, the experimentally observed decrease in the k/K in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k, and the increase in K.