The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.
Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D003979:Diazonium Compounds; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D046911:Macromolecular Substances; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D003979:Diazonium Compounds; D046911:Macromolecular Substances; D006639:Histidine; D000469:Alkaline Phosphatase; D015032:Zinc