Formamide-induced dissociation and inactivation of Escherichia coli alkaline phosphatase. Metal-dependent reassociation and restoration of activity from isolated subunits.

Abstract

Alkaline phosphatase from Escherichia coli has been reversibly dissociated by treatment with low concentrations of formamide. The monomer retains the capacity to bind metals and to regenerate catalytically active dimer that is identical with the native dimeric enzyme. The rate and extent of dissociation of dimer to monomer depend upon pH, ionic strength, temperature, formamide concentration, and enzyme-bound metal. Under appropriate experimental conditions, reassociation can be greatly slowed, allowing the properties of the monomer to be examined in solution. The formamide-induced apo monomer has a conformation distinct from that of the dimer and zinc- or cobalt-containing monomers. The monomer tightly binds 1 mol of zinc or cobalt in a metal-binding site altered from those of the dimer but is catalytically inactive. pH, ionic strength, and formamide concentration all influence reassociation. Hydrophobic forces are implicated as important in subunit interactions. The effect of metal content on the dissociation--reassociation process underscores the essential role that metals play in maintaining enzyme tertiary structure and reveals a new role in stabilizing the quaternary structure.