The mechanism of hydrolysis of the nitrile (N-acetyl-phenylalanyl-2-amino-propionitrile, I) catalyzed by Gln19Glu mutant of papain has been studied by nanosecond molecular dynamics (MD) simulations. MD simulations of the complex of mutant enzyme with I and of mutant enzyme covalently attached to both neutral (11) and protonated (111) thioimidate intermediates were performed. An MD simulation with the wildtype enzyme-I complex was undertaken as a reference. The ion pair between protonated His159 and thiolate of Cys25 is coplanar, and the hydrogen bonding interaction S-(25)(...)HD1-ND1 (159) is observed throughout MD simulation of the mutant enzyme-I complex. Such a sustained hydrogen bond is absent in nitrile-bound wild-type papain due to the flexibility of the imidazole ring of His159. The nature of the residue at position 19 plays a critical role in the hydrolysis of the covalent thioimidate intermediate. When position 19 represents Glu, the imidazolium ion of His159-ND1(+...)Cys25-S- ion pair is distant, on average, from the nitrile nitrogen of substrate I. Near attack conformers (NACs) have been identified in which His159-ImH(+) is positioned to initiate a general acid-catalyzed addition of Cys-S- to nitrile. Though Glu19-CO2H is distant from nitrile nitrogen in the mutant-I structure, MD simulations of the mutant-Ill covalent adduct finds Glu19-CO2H hydrogen bonded to the thioimide nitrogen of Ill. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19-CO2- With mutant-bound protonated thioimidate (111). This observation supports Glu19-CO2H general acid catalysis of the formation of mutant-Ill. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.