Evidence for histidyl residues at the Zn2+ binding sites of monomeric and dimeric forms of alkaline phosphatase.
Chemical modification of Escherichia coli alkaline phosphatase using the group-specific reagent, ethoxyformic anhydride, has demonstrated that 3 histidyl residues/subunit are modified with a concomitant loss of enzyme activity. Reaction with [14C]ethoxyformic anhydride indicates that only three ethoxyformyl groups are incorporated per subunit, confirming that no other amino acid residues are modified under these conditions. Zinc ions protect alkaline phosphatase from inactivation as well as from histidine modification, thus implicating all 3 histidyl residues in Zn2+ binding. The ethoxyformylation reaction was also used to characterize Zn2+ binding sites in immobilized dimeric and monomeric alkaline phosphatase derivatives. The immobilized dimeric alkaline phosphatase was inactivated with ethoxyformic anhydride at a rate similar to that of the soluble enzyme, demonstrating that immobilization did not significantly alter the chemical environment of the Zn2+ binding site. The catalytically inactive, immobilized monomer of alkaline phosphatase was modified more rapidly with ethoxyformic anhydride, demonstrated by the loss of its ability to form functionally active enzyme upon titration with nascent soluble subunits. Moreover, Zn2+ protects the immobilized subunit alkaline phosphatase against this modification, indicating that the isolated subunits of alkaline phosphatase bind Zn2+. These results are consistent with a model for renaturation of the dimeric enzyme in which individual subunits refold and bind Zn2+ before which individual subunits refold and bind Zn2+ before establishing subunit interactions to regain catalytic activity.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D046911:Macromolecular Substances; D006639:Histidine; D000469:Alkaline Phosphatase; D015032:Zinc