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Titolo:
Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus
Autore:
Cacciapuoti, G; Servillo, L; Moretti, MA; Porcelli, M;
Indirizzi:
Univ Naples 2, Dipartimento Biochim & Biofis F Cedrangolo, I-80138 Naples,Italy Univ Naples 2 Naples Italy I-80138 is F Cedrangolo, I-80138 Naples,Italy
Titolo Testata:
EXTREMOPHILES
fascicolo: 5, volume: 5, anno: 2001,
pagine: 295 - 302
SICI:
1431-0651(200110)5:5<295:CCASIB>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
PURINE NUCLEOSIDE PHOSPHORYLASE; ESCHERICHIA-COLI; DISULFIDE BONDS; SALMONELLA-TYPHIMURIUM; CRYSTAL-STRUCTURE; PROTEIN; GENE; PURIFICATION; MECHANISM; SEQUENCE;
Keywords:
5 '-methylthioadenosine phosphorylase; Sulfolobus solfataricus; protein stability; substrate-induced conformational change; limited proteolysis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
20
Recensione:
Indirizzi per estratti:
Indirizzo: Cacciapuoti, G Univ Naples 2, Dipartimento Biochim & Biofis F Cedrangolo, Via Costantinopoli 16, I-80138 Naples, Italy Univ Naples 2 Via Costantinopoli 16 Naples Italy I-80138 y
Citazione:
G. Cacciapuoti et al., "Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus", EXTREMOPHIL, 5(5), 2001, pp. 295-302

Abstract

The effect of phosphate, its analogues, and other substrates on structuralfeatures 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 (T-m) of recombinant SsMTAP increased from 111 degrees to 118 degreesC; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degreesC 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 previouslyaccessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

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Documento generato il 03/07/20 alle ore 00:45:31