Naslov
Alkaline phosphatase from E. coli - metal ion dependent conformational changes

Autori
Bučević-Popović, Viljemka ; Orhanović, Stjepan ; Pavela-Vrančič, Maja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
The 15th Dubrovnik International Course & Conference on the Interfaces among Mathematics, Chemistry and Computer Sciences : Math/Chem/Comp 2000 : Book of Abstracts / Graovac, Ante ; Plavšić Dejan ; Pokrić Biserka ; Smrečki Vilko - Dubrovnik, 2000, 11-11

Skup
Dubrovnik International Course and Conference on the Interfaces among Mathematics, Chemistry and Computer Sciences (15 ; 2000)

Mjesto i datum
Dubrovnik, Hrvatska, 19.06.2000. - 24.06.2000

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
alkaline phosphatase; conformational changes

Sažetak
Alkaline phosphatase is a multimeric metalloprotein catalyzing phosphohydrolytic and phosphoryl transfer reactions. It is present in the periplasmatic space of prokaryotes or associated with the outer side of the plasmatic membrane in eukaryotes, suggesting its involvement in phosphate transport into the cell. According to the crystallographic study, alkaline phosphatase from E. coli (PhoA) is composed of two apparently identical subunits. Three classes of metal binding sites exist in active site on each subunit, two of which bind Zn and one Mg ion. A vast amount of data exists indicating a non-equivalence of the PhoA subunits, including Pi binding with strong negative cooperativity, biphasic thermal inactivation and deviations from Michaelis-Menten kinetics substantiated by enzyme asymmetry. Unequal saturation of the subunits with metal ions could induce the observed enzyme asymmetry. We have used limited proteolysis as a probe of conformation to examine the possible structural differences between the subunits of alkaline phosphatase. Susceptibility of the enzyme to digestion with proteinase K or trypsin was studied in solutions containing different concentrations of Zn and Mg. The progress of the proteolytic reaction was monitored by PhoA activity assay and SDS-PAGE. The proteolytic pattern and the time course of digestion were dependent on the concentration of metal ions in the reaction solution. Metal-free alkaline phosphatase was highly susceptible to proteolysis, while the presence of bound metals provided protection form digestion under the same reaction conditions. We conclude that binding of metal ions to alkaline phosphatase induces conformational changes in the protein that alter its susceptibility to proteolysis and influence its enzymatic activity.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA