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Finally, to determine whether 2,3-BPG has an inhibitory effect on PNP activation by competing for binding to these three amino acid residues in the active site of PNP identified by molecular docking and molecular dynamics simulations, we mutated Ser33 and His86 of PNP to alanine and Ala116 to phenylalanine, after which we performed protein expression experiments in HEK293T cells (Fig