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Activation of an Asp‐124→Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine
Author(s) -
Pries Frens,
Kingma Jaap,
Janssen Dick B.
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01420-6
Subject(s) - deamidation , chemistry , asparagine , stereochemistry , residue (chemistry) , nucleophile , dehalogenase , active site , enzyme , biochemistry , hydrolysis , phosphofructokinase 2 , site directed mutagenesis , mutant , catalysis , gene
Haloalkane dehalogenase hydrolyses various 1‐halo‐ n ‐alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl‐enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp‐124) that displaces the halogen during formation of the intermediate was changed to an amide by site‐directed mutagenesis (Asp‐124→Asn). Activity measurements and analysis of peptides containing the nucleophilic residue showed that the mutant enzyme was inactive, but that the activity increased by rapid deamidation of the asparagine residue, yielding wild type enzyme. There was no indication for isoaspartate formation during this process. The results suggest that a water molecule that is located close to the carboxyl function of Asp‐124 in the X‐ray structure is highly reactive and is responsible for the observed deamidation.