Stability of a thermophilic TIM-barrel enzyme: indoleglycerol-phosphate synthase from the thermophilic arcaeon Sulfolobus solfataricus
Articolo
Data di Pubblicazione:
1997
Citazione:
Stability of a thermophilic TIM-barrel enzyme: indoleglycerol-phosphate synthase from the thermophilic arcaeon Sulfolobus solfataricus / G. Andreotti, M.V. Cubellis, M. di Palo, D. Fessas, G. Sannia, G. Marino. - In: BIOCHEMICAL JOURNAL. - ISSN 0264-6021. - 323:1(1997), pp. 259-264. [10.1042/bj3230259]
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 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 k(cat) 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.
Tipologia IRIS:
01 - Articolo su periodico
Elenco autori:
G. Andreotti, M.V. Cubellis, M. di Palo, D. Fessas, G. Sannia, G. Marino
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