Activation of an Asp-124-->Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine.

Abstract:

: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.

journal_name

FEBS Lett

journal_title

FEBS letters

authors

Pries F,Kingma J,Janssen DB

doi

10.1016/0014-5793(94)01420-6

subject

Has Abstract

pub_date

1995-01-23 00:00:00

pages

171-4

issue

2

eissn

0014-5793

issn

1873-3468

pii

0014-5793(94)01420-6

journal_volume

358

pub_type

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