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 fro m Sulfolobus solfataricus were studied as a function of pH and tempera ture. In this paper we focus on three points: (1) the longterm stabili ty of the protein to irreversible denaturation at high temperature; (2 ) the short-term stability of the protein to reversible temperature-dr iven unfolding; and (3) the dependence of its activity on temperature, Results can be summarized as follows: (a) the same first-order kineti c constant (0.020 +/- 0.003 min(-1)) was determined at different pH va lues (6.5, 8.0 and 9.5) from long-term stability experiments at 80 deg rees C; (b) short-term stability experiments revealed different behavi our 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 k(cat) values at pH 9.0. We also investigated the stability in the presence o f 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 a t 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 th e 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.