Determination by Cadmium-113 Nuclear Magnetic Resonance of the Structural Basis for Metal Ion Dependent Anticooperativity in Alkaline Phosphatase

Cadmium-113 nuclear magnetic resonance (¹¹³Cd NMR) has been used to probe the binding characteristics of¹¹³Cd²⁺ to the three classes of metal binding sites in Escherichia coli alkaline phosphatase to help elucidate the molecular origin of themetal ion dependent “half-sites” reactivity exhibited by this dimeric Zn²⁺metalloenzyme [Otvos, J. D., Armitage, I. M., Chlebowski, J. F., & Coleman, J. E. (1979) J. Biol. Chem. 254, 4707-4713], in the absence of phosphate, the first two¹¹³Cd²⁺ions added to the apodimer give rise to a single¹¹³Cd²⁺resonance (169 ppm), indicating selective binding to the pair of symmetrically disposed A sites. Resonances arising from additional¹¹³Cd²⁺bound to the B and C sites cannot be observed; B- and/or C-site occupation also renders the A-site¹¹³Cd resonance undetectable. Both these observations have been attributed to severe chemical exchange broadening in the A-, B-, and C-site¹¹³Cd signals induced by an unknown modulation process(es). Interestingly, covalent phosphorylation of the active-site serine residues abolishes this exchange modulation, allowing three separate resonances to be detected and assigned to¹¹³Cd²⁺located at each of the three classes of metal binding sites in the enzyme. By varying the metal composition of the phosphorylated enzyme, we have characterized the correlations that exist between the chemical shifts and intensities of these¹¹³Cd resonances and the metal occupancies of the A, B, and C sites in the individual subunits. This information has allowed us to conclude that the half-sites phosphorylation of the Cd²⁺enzyme is accompanied by a slow migration of half the Cd²⁺originally located at the A sites to the B sites on the phosphorylated subunits. The driving force for this metal redistribution, which at equilibrium leaves half the subunits devoid of metal ion and thereby incapable of binding phosphate, is apparently the dramatic stabilization of the complex of Cd²⁺with the B sites, which was demonstrated to occur in those subunits that become phosphorylated. From the kinetics of both phosphorylation and metal redistribution in Cd²⁺enzyme, we suggest that population of the A and B sites in a subunit, rather than the A site alone, constitutes the minimum requirement for induction of catalytic function in alkaline phosphatase. The spin relaxation properties of the enzyme-bound¹¹³Cd²⁺ions are also briefly discussed.