Stability of a thermophilic TIM-barrel enzyme: indole-3-glycerol phosphate synthase from the thermophilic archaeon Sulfolobus solfataricus.
The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points: (1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature. Results can be summarized as follows: (a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.
Andreotti
G
G
Dipartimento di Chimica Organica e Biologica, Via Mezzocannone 16, Universit à di Napoli 'Federico II', 80134 Napoli, Italy.
Cubellis
M V
MV
Palo
M D
MD
Fessas
D
D
Sannia
G
G
Marino
G
G
D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016944:Sulfolobus / Q000201:enzymology*
D006146:Guanidines; D011994:Recombinant Proteins; D007209:Indole-3-Glycerol-Phosphate Synthase; D019791:Guanidine