ACTIVATION OF AN ASP-124-]ASN MUTANT OF HALOALKANE DEHALOGENASE BY HYDROLYTIC DEAMIDATION OF ASPARAGINE

Haloalkane dehalogenase hydrolyses various 1-halon-alkanes to the corr esponding alcohols by covalent catalysis with formation of an alkyl-en zyme intermediate. The carboxylate function of the nucleophilic aspart ate (Asp-124) that displaces the halogen during formation of the inter mediate 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 t hat the activity increased by rapid deamidation of the asparagine resi due, yielding wild type enzyme. There was no indication for isoasparta te formation during this process. The results suggest that a water mol ecule that is located close to the carboxyl function of Asp-124 in the X-ray structure is highly reactive and is responsible for the observe d deamidation.