In this paper, elevated pressures up to 750 atm
(1 atm = 101 kPa) were found to have a strong stabilizing
effect on two extremely thermophilic glutamate dehydrogenases
(GDHs): the native enzyme from the hyperthermophile Pyrococcus
furiosus (Pf), and a recombinant GDH mutant containing
an extra tetrapeptide at the C-terminus (rGDHt).
The presence of the tetrapeptide greatly destabilized the
recombinant mutant at ambient pressure; however, the destabilizing
effect was largely reversed by the application of pressure.
Electron spin resonance (ESR) spectroscopy of a spin-label
attached to the terminal cysteine of rGDHt
revealed a high degree of mobility, suggesting that destabilization
is due to weakened intersubunit ion-pair interactions induced
by thermal fluctuations of the tetrapeptide. For both enzymes,
the stabilizing effect of pressure increased with temperature
as well as pressure, reaching 36-fold for rGDHt
at 105 °C and 750 atm, the largest pressure-induced
thermostabilization of an enzyme reported to date. Stabilization
of both native GDH and rGDHt was also
achieved by adding glycerol. Based on the kinetics of thermal
inactivation and the known effects of glycerol on protein
structure, a mechanism of pressure-induced thermostabilization
is proposed.