Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus

In this paper, elevated pressures up to 750 arm (1 atm = 101 kPa) were foun d to have a strong stabilizing effect on two extremely thermophilic glutama te dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyroc occus furiosus (PS), and a recombinant GDH mutant containing an extra tetra peptide at the C-terminus (rGDH(t)). The presence of the tetrapeptide great ly destabilized the recombinant mutant at ambient pressure; however, the de stabilizing effect was largely reversed by the application of pressure. Ele ctron spin resonance (ESR) spectroscopy of a spin-label attached to the ter minal cysteine of rGDH(t) revealed a high degree of mobility, suggesting th at destabilization is due to weakened intersubunit ion-pair interactions in duced by thermal fluctuations of the tetrapeptide. For both enzymes, the st abilizing effect of pressure increased with temperature as well as pressure , reaching 36-fold for rGDH(t) at 105 degrees C and 750 atm, the largest pr essure-induced thermostabilization of an enzyme reported to date. Stabiliza tion of both native GDH and rGDH(t) was also achieved by adding glycerol. B ased on the kinetics of thermal inactivation and the known effects of glyce rol on protein structure, a mechanism of pressure-induced thermostabilizati on is proposed.