Chemical rescue at the catalytically disabled clostridial glutamate dehydrogenase mutant D165S by fluoride ion

The catalytically disabled Asp(165) --> Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzy me in a standard glutamate oxidation assay and 1000-fold less activity in t he reductive-amination reaction. The large reduction in the rate has been a ttributed 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 inh ibiting WT activity by 20-fold in the forward reaction. For the reverse rea ction, F- (1 M) activates the mutant 4-fold and inhibits WT activity to app rox. 64 %. The net result when 1 M F- is present is a decrease in the WT:mu tant activity ratio from 100 000 to 5 for the forward reaction. None of the other halides tested, nor NO3-, CHCOO- or HCOO-, give comparable activatio n. Re-activation took 15-30 s under assay conditions, suggesting the possib ility of conformational change; CD spectroscopy, however, provided no evide nce of a substantial change and kinetics of modification using 5,5'-dithiob is(2-nitrobenzoic acid) suggested only subtle structural rearrangement. Thi s phenomenon is discussed in the light of available information about the s tructure of the mutant enzyme. It is suggested that the F- ion provides a f ixed negative charge at the position of the missing aspartate carboxyl grou p. Therefore, this appears to be an example of 'chemical rescue'.