Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N.
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.
Paradisi
Francesca
F
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
Woolfson
Ruth
R
Geoghegan
Kieran F
KF
Engel
Paul C
PC
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D016296:Mutagenesis; D017510:Protein Folding
D017931:DNA Primers; D005969:Glutamate Dehydrogenase