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Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N
Author(s) -
Paradisi Francesca,
Woolfson Ruth,
Geoghegan Kieran F.,
Engel Paul C.
Publication year - 2005
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/j.febslet.2005.03.098
Subject(s) - deamidation , mutant , residue (chemistry) , chemistry , biochemistry , enzyme , glutamate receptor , glutamate dehydrogenase , microbiology and biotechnology , biology , gene , receptor
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.