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 longterm 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 ®rst-order kinetic constant (0.020³0.003 min-") was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 °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 °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 ®tting of the equilibrium unfolding transition obtained by ¯uorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 °C the folded conformation is more stable than the unfolded conformation by (14.7³1.2) kJ}mol at pH 7.0 and by (25.5³1.8) kJ}mol at pH 9.0.
Stability of a thermophilic TIM-barrel enzyme: indole-3-glycerol phosphate synthase from the thermophilic archaeon Sulfolobus solfataricus
G Andreotti;
1997
Abstract
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 longterm 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 ®rst-order kinetic constant (0.020³0.003 min-") was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 °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 °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 ®tting of the equilibrium unfolding transition obtained by ¯uorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 °C the folded conformation is more stable than the unfolded conformation by (14.7³1.2) kJ}mol at pH 7.0 and by (25.5³1.8) kJ}mol at pH 9.0.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
