title: Protein engineering of nitrile hydratase activity of papain: molecular dynamics study of a mutant and wild-type enzyme.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Santa Barbara, Santa Barbara, California 93106, USA.
authors.forename: Swarnalatha Y
authors.initials: SY
authors.lastname: Reddy
authors.forename: Kalju
authors.initials: K
authors.lastname: Kahn
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Zheng
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Bruice
mesh_terms: D003198:Computer Simulation; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009570:Nitriles; D010206:Papain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives; D011487:Protein Conformation; D015202:Protein Engineering; D013816:Thermodynamics
chemical_list: C470327:N-acetyl-phenylalanyl-2-amino-propionitrile; D009570:Nitriles; D010649:Phenylalanine; D010206:Papain; D006836:Hydro-Lyases; C048831:nitrile hydratase