American Chemical Society
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Protein Engineering of Nitrile Hydratase Activity of Papain:  Molecular Dynamics Study of a Mutant and Wild-Type Enzyme

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journal contribution
posted on 2002-10-09, 00:00 authored by Swarnalatha Y. Reddy, Kalju Kahn, Ya-Jun Zheng, Thomas C. Bruice
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−CO2H is distant from nitrile nitrogen in the mutant·I structure, MD simulations of the mutant·II covalent adduct finds Glu19−CO2H hydrogen bonded to the thioimide nitrogen of II. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19−CO2- with mutant-bound protonated thioimidate (III). This observation supports Glu19−CO2H general acid catalysis of the formation of mutant·III. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.