engleski

Metal ion dependent conformational changes of alkaline phosphatase

Alkaline phosphatase (PhoA) 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 as a possible biological role of alkaline phosphatases. According to the crystallographic study, PhoA from E. coli is composed of two apparently identical subunits. On the other hand, a vast amount of data exists indicating a non-equivalence of the PhoA subunits, including Pi binding with strong negative cooperativity, unequal saturation with Mg2+, biphasic thermal inactivation and deviations from Michaelis-Menten kinetics substantiated by enzyme asymmetry. These data suggest conformational heterogeneity between the subunits. It has been reported that PhoA from E. coli is not fully saturated with metal ions. Unequal saturation of the subunits could induce the observed enzyme asymmetry. The dependence of the kinetic constants and the deviations from Michaelis-Menten kinetics on the Mg2+-ion concentration has been investigated. We have examined the possible structural differences between the subunits using limited proteolysis with trypsin as a probe of conformational changes. Susceptibility to proteolysis was studied in solutions containing different concentrations of metal ions and a metal-complexing agent. A variable kinetic behaviour of PhoA was observed. The progress of the proteolytic reaction was monitored by SDS-PAGE and the PhoA activity assay. The proteolytic pattern and the time course of tryptric digestion were dependent on the concentration of metal ions in the reaction solution. We conclude that binding of metal ions to PhoA induces conformational changes in the protein that alter its susceptibility to proteolysis and influence its enzymatic activity.

alkaline phosphatase; conformational changes; metal ion

nije evidentirano