Chemical rescue of the catalytically disabled clostridial glutamate dehydrogenase mutant D165S by fluoride ion.
The catalytically disabled Asp165-->Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzyme in a standard glutamate oxidation assay and 1000-fold less activity in the reductive-amination reaction. The large reduction in the rate has been attributed to removal of the negative charge and the postulated proton-donor capacity of the aspartate carboxyl group. However, fluoride ion (1 M NaF) causes a 1000-fold activation of the mutant enzyme while simultaneously inhibiting WT activity by 20-fold in the forward reaction. For the reverse reaction, F- (1 M) activates the mutant 4-fold and inhibits WT activity to approx. 64%. The net result when 1 M F- is present is a decrease in the WT:mutant activity ratio from 100000 to 5 for the forward reaction. None of the other halides tested, nor NO3(-), CHCOO- or HCOO-, give comparable activation. Re-activation took 15-30 s under assay conditions, suggesting the possibility of conformational change; CD spectroscopy, however, provided no evidence of a substantial change and kinetics of modification using 5,5'-dithiobis(2-nitrobenzoic acid) suggested only subtle structural rearrangement. This phenomenon is discussed in the light of available information about the structure of the mutant enzyme. It is suggested that the F- ion provides a fixed negative charge at the position of the missing aspartate carboxyl group. Therefore, this appears to be an example of 'chemical rescue'.
Hayden
B M
BM
Department of Biochemistry, University College Dublin, Merville House, Belfield, Dublin 4, Ireland.
Dean
J L
JL
Martin
S R
SR
Engel
P C
PC
D002384:Catalysis; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D004228:Dithionitrobenzoic Acid; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012969:Sodium Fluoride / Q000737:chemistry*
D011994:Recombinant Proteins; D012969:Sodium Fluoride; D004228:Dithionitrobenzoic Acid; D005969:Glutamate Dehydrogenase