engleski

Mass spectrometric analysis of metal ion induced conformational changes in alkaline phosphatase from E. coli

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. It has previously been established that the binding of Zn2+ is required for both catalysis and structural stabilization while Mg2+ is not essential, but is required for full stabilization and activation of the enzyme. AP displays significant structural changes during metal-ion binding, supporting cooperative interactions between the subunits of the dimeric enzyme. Here, we present data on the dynamic properties of AP and characterize structural changes that accompany variations in metal-ion content. Apo-AP and AP, reconstituted at various Zn2+ and/or Mg2+ to dimer ratios, were submitted to limited proteolysis with trypsin. MALDI-TOF mass spectrometry was employed to follow the time course of proteolysis, to establish the sites of cleavage, and to identify the segments of the polypeptide chain undergoing major conformational changes induced by metal-ion binding. Analysis of the proteolytic pattern revealed an internal cleavage site at Arg-293 site, residing in a highly flexible region of the polypeptide chain, reflecting a position of conformational flexibility essential for catalysis. A specific shielding of a region distant from the metal-binding site has been demonstrated, implying transmission of conformational changes, induced by metal-ion binding to the adjacent subunit, across the subunit interface.

alkaline phosphatase; limited proteolysis; MALDI-TOF mass spectrometry

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