title: Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.
abstract: 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 degrees 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.
authors.affiliation: Department of Chemical Engineering, University of California, Berkeley 94720, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Sun
authors.forename: N
authors.initials: N
authors.lastname: Tolliday
authors.forename: C
authors.initials: C
authors.lastname: Vetriani
authors.forename: F T
authors.initials: FT
authors.lastname: Robb
authors.forename: D S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D005990:Glycerol / Q000494:pharmacology; D008958:Models, Molecular; D011312:Pressure; D011489:Protein Denaturation; D020027:Pyrococcus furiosus / Q000737:chemistry*; D013113:Spin Labels; D013696:Temperature; D013997:Time Factors
chemical_list: D013113:Spin Labels; D005969:Glutamate Dehydrogenase; D005990:Glycerol