Conformational changes and stabilization induced by phosphate binding to 5′-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus.

Cacciapuoti, Giovanna; Servillo, Luigi; Moretti, Maria; Porcelli, Marina

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5′-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111° to 118°C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90°C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

NITROGEN excretion; PROTEOLYTIC enzymes; FLUORESCENCE spectroscopy; SPECTRUM analysis; METABOLISM; PROTEOLYSIS; PROTEIN metabolism

Extremophiles, 2001, Vol 5, Issue 5, p295

1431-0651

Academic Journal

10.1007/s007920100205