Activation of an Asp‐124→Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine

FEBS Letters

Kingma, Jaap¹  Janssen, Dick B.¹  Pries, Frens¹ 

[1] Department of Biochemistry, Groningen Biomolecular and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

关键词: Dehalogenase;    Nucleophilic substitution;    Deamidation;    Active site mutant;    Reactivation;    Asparagine;   

DOI  :  10.1016/0014-5793(94)01420-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown