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

Published online by Cambridge University Press:  01 May 1999

MICHAEL M.C. SUN ,
NICOLA TOLLIDAY ,
COSTANTINO VETRIANI ,
FRANK T. ROBB  and
DOUGLAS S. CLARK
MICHAEL M.C. SUN
Affiliation:
Department of Chemical Engineering, University of California, Berkeley, California 94720
NICOLA TOLLIDAY
Affiliation:
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
COSTANTINO VETRIANI
Affiliation:
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
FRANK T. ROBB
Affiliation:
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
DOUGLAS S. CLARK
Affiliation:
Department of Chemical Engineering, University of California, Berkeley, California 94720
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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 °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.

Keywords

electron spin resonanceglutamate dehydrogenaseglycerolpressurePyrococcus furiosusstabilitytemperature
Type
Research Article
Information
Protein Science , Volume 8 , Issue 5 , May 1999 , pp. 1056 - 1063
Copyright
1999 The Protein Society

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