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- Title
Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N
- Authors
Paradisi, Francesca; Woolfson, Ruth; Geoghegan, Kieran F.; Engel, Paul C.
- Abstract
Abstract: Previously a mutant of clostridial glutamate dehydrogenase with the catalytic Asp-165 replaced by Asn was shown to regain activity through spontaneous, specific deamidation of this residue. A double mutant D165N/K125A has now been constructed, in which the catalytic Lys is also replaced. This was successfully over-expressed and according to several criteria appears to be correctly folded. The double mutant was incubated for 35 days under conditions where D165N reactivates. LC–MS analysis of tryptic digests of timed samples showed no significant deamidation. This confirms that the reactivation of D165N is a consequence of the catalytic chemistry of the enzyme’s active site.
- Subjects
DEHYDROGENASES; SURFACE chemistry; GENETIC mutation; RADIOGENETICS
- Publication
FEBS Letters, 2005, Vol 579, Issue 13, p2830
- ISSN
0014-5793
- Publication type
Article
- DOI
10.1016/j.febslet.2005.03.098