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

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobu s solfataricus (SsMTAP) was investigated. Phosphate was found to exert a si gnificant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzyme s. In the presence of 100 mM phosphate: (i) the apparent transition tempera ture (T-m) of recombinant SsMTAP increased from 111 degrees to 118 degreesC ; and (ii) the enzyme still retained 40% and 30% activity, respectively, af ter 30 min of incubation at 90 degreesC with 2% SDS or 8 M urea. The struct ure modification of SsMTAP by phosphate binding was probed by limited prote olysis with subtilisin and proteinase K and analysis of polypeptide fragmen ts by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP aga inst protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal r egion of the enzyme. The conformational changes of SsMTAP induced by phosph ate and ribose-1phosphate were analyzed by fluorescence spectroscopy, and m odifications of the protein intrinsic fluorophore exposure, as a consequenc e of substrate binding, were evidenced.