engleski

Alkaline phosphatase conformational changes assesed by limited proteolysis and MALDI-TOF mass spectrometry

Alkaline phosphatase (AP ; E.C. 3.1.3.1.) from Escherichia coli is a homodimeric metalloenzyme containing two Zn2+ and one Mg2+ binding site in each active center. AP displays deviations from Michaelis-Menten kinetics, that have been interpreted in terms of negative cooperativity or described by the model of a dimeric enzyme with unequal subunits. It has been postulated that conformational changes induced by binding of metal ions might be responsible for the expression of negative cooperativity or the observed enzyme assymetry. In this study, metal-ion induced conformational changes of AP were examined by limited proteolysis with trypsin and MALDI-TOF mass spectrometry. A marked decrease in susceptibility toward proteolytic degradation was observed upon addition of Zn2+ to apo-AP. The addition of Mg2+ together with Zn2+ further enhanced the resistance to tryptic digestion, while Mg2+alone had no effect. Analysis of AP tryptic fragments by MALDI-TOF mass spectrometry revealed differences in proteolytic susceptibility at the Arg-293 cleavage site. The results suggest that the binding of metal ions evokes conformational changes that extend far from the metal binding site.

alkaline phosphatase; limited proteolysis; MALDI-TOF mass spectrometry

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