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 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 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees 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.
Cacciapuoti
G
G
Dipartimento di Biochimica e Biofisica "F Cedrangolo," Seconda Universita degli Studi di Napoli, Via Costantinopoli 16, 80138, Naples, Italy. Giovanna.CACCIAPUOTI@unina2.it
Servillo
L
L
Moretti
M A
MA
Porcelli
M
M
D001665:Binding Sites; D010450:Endopeptidases; D004795:Enzyme Stability; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature; D014508:Urea
D010710:Phosphates; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D010450:Endopeptidases