Engineering nitrile hydratase activity into a cysteine protease by a single mutation.
A peptide nitrile hydratase activity has been engineered into the cysteine protease papain by a single carefully selected mutation at the active site of the enzyme. The papain variant Gln19Glu hydrolyzes the substrate MeOCO-PheAla-CN to the corresponding amide with a kcat/KM value of 1.15 x 10(3) M-1 s-1. The reaction leads to an accumulation of the corresponding amide, which is then further hydrolyzed to the acid by the natural amidase activity of the enzyme. The pH-dependency of the nitrile hydratase activity of Gln19Glu supports the involvement of the acid form of the Glu19 residue in the reaction. The wild type enzyme displays very weak nitrile hydratase activity, and the introduction of a glutamic acid residue in the oxyanion hole of papain causes the kcat at pH 5 to increase by a factor of at least 4 x 10(5). Peptide nitriles react with cysteine proteases to form thioimidates, and the role of the glutamic acid residue introduced at position 19 in the Gln19Glu enzyme is to participate in the acid-catalyzed hydrolysis of the thiomidate to the amide by the provision of a proton to form the more reactive protonated thioimidate. This dramatically decreases the energy barrier for the hydrolysis of the thioimidate, as shown by the impressive increase in kcat. The success of the rational approach undertaken is a consequence of the level of understanding of the basic catalytic properties of cysteine proteases of the papain family.
Dufour
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E
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Storer
A C
AC
Ménard
R
R
D000581:Amidohydrolases / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000378:metabolism*; D010206:Papain / Q000235:genetics*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D009570:Nitriles; D010455:Peptides; D010206:Papain; D000581:Amidohydrolases; D006836:Hydro-Lyases; C048831:nitrile hydratase