Allosteric Interactions between Metal Ion and Phosphate at the Active Sites of Alkaline Phosphatase as Determined by 31P NMR and l13Cd NMR*

““Cd and :“P NMR have been used to determine the effect of variations in the nature and stoichiometry of bound metal ion and pH on the allosteric interactions (negative cooperativity) induced on association of phosphate with the dimeric zinc metalloenzyme Escherichia coli alkaline phosphatase. At pH 8.0, successive additions of an extra two Zn’+ ions and one Mg”+ ion to the Znzs+ enzyme result in structural alterations of the noncovalent phosphate complex (E. P) reflected in a progressive upfield chemical shift of the :“P resonance from 5.1 ppm (Zngz+ enzyme) to 4.2 and 3.5 ppm, respectively. Variation of metal ion content does not alter the stoichiometry of tight phosphate binding; in all cases a single phosphate ligand is bound per protein dimer. Phosphate binding to the CoL2+ enzyme at pH 8.0 (where tightly bound and free P, exist under slow exchange conditions) and pH 6.5 (where Pi is in fast exchange) are similarly consistent with the tight association of one Pi/ dimer. Observation of the NMR of both the ligand and metal ion nuclei in the covalent phosphate complex (E .P) of the Cd,?+ enzyme at pH 6.5 confirms these results. Two equivalents of Cd”+/dimer are required to generate the maximum of 1 eq of phosphoryl enzymeldimer, observed as a characteristic low field (-8.0 ppm) resonance in the n’P NMR spectrum. The “Wd NMR spectrum of the unliganded enzyme shows the Cd”+ ions to exist in identical environments, since a single resonance is present 170 ppm downfield from the standard, 0.1 M CdCIO,. Covalent phosphorylation at a single site results in appearance of two resonances of equal intensity at 142 and 55 ppm. The presence of 1 mol of excess phosphate does not alter the stoichiometry of phosphoryl enzyme formation or the altered environments of the metal ions. These results are consistent only with the existence of negative homotropic interactions between the subunits induced on ligand (phosphate) binding resulting in