Abstract
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 (II) and protonated (III) thioimidate intermediates were performed. An MD simulation with the wild-type 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-CO(2)H is distant from nitrile nitrogen in the mutant.I structure, MD simulations of the mutant.II covalent adduct finds Glu19-CO(2)H hydrogen bonded to the thioimide nitrogen of II. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19-CO(2)(-) with mutant-bound protonated thioimidate (III). This observation supports Glu19-CO(2)H general acid catalysis of the formation of mutant.III. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have