after incubation with chymotrypsin enzyme loses 50% activity within 30 min and a combination of 2-oxoglutarate and NADP+ affords near complete protection against chymotrypsin
elevated pressures up to 750 atm have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases: the native enzyme from the hyperthermophile Pyrococcus furiosus, and a recombinant mutant enzyme containing an extra tetrapeptide at the C-terminus. The presence of the tetrapeptide greatly destabilizes the recombinant mutant at ambient pressure; however, the destabilizing effect is largely reversed by the application of pressure. Destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increases with temperature as well as pressure, reaching 36fold for recombinant enzyme at 105°C and 750 atm
Gdh3-encoded enzyme undergoes in vitro deamination at a particular asparaginyl residue which is absent in homologous isoenzyme, Gdh1p. Deamination of Asn54 is observed in vitro when Gdh3p is incubated at alkaline pH. The specific deamination of Asn54, could account, in part, for the relative lower stability of the GDH3-encoded protein
in presence of polyethyleneimine , the enzyme almost maintains the full initial activity after 2 h under conditions where the untreated enzyme retains only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappears. This stabilization is maintained in the pH range 59, but it is lost at high ionic strength.
in the presence of 4 M NaCl the purified enzyme remains fully active at room temperature for months. The stability is rapidly lost upon lowering the salt concentration
the purified enzyme can withstand at least three freezing (-20°C) and thawing (25°C) cycles without significant effects on activity the enzyme can be lyophilzed and stored for several months without loss of activity
the stability of this enzyme is increased in the presence of Li+ in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate, and a very significant dependence of the enzyme stability on protein concentration is found
wild-type enzyme and mutant enzymes D167T and T138E are thermostabilized, although to different degrees, by the application of 500 atm. The degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167