Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 μM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6.kJ mol-1, which is considerably lower than that of the holoenzyme (494.4kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80°C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.

Villa, A., Zecca, L., Fusi, P., Colombo, S., Tedeschi, G., & Tortora, P. (1993). Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus. BIOCHEMICAL JOURNAL, 295, 827-831.

Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus

FUSI, PAOLA ALESSANDRA;COLOMBO, SONIA;TORTORA, PAOLO
1993

Abstract

Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 μM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6.kJ mol-1, which is considerably lower than that of the holoenzyme (494.4kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80°C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.
Articolo in rivista - Articolo scientifico
Archaebacteria,Themostable enzyme,Solfolobus solfataricus
English
827
831
Villa, A., Zecca, L., Fusi, P., Colombo, S., Tedeschi, G., & Tortora, P. (1993). Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus. BIOCHEMICAL JOURNAL, 295, 827-831.
Villa, A; Zecca, L; Fusi, P; Colombo, S; Tedeschi, G; Tortora, P
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/12451
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