31P nuclear magnetic resonance of phosphoenzyme intermediates of alkaline phosphatase.

Covalent (E-P) and noncovalent (E X P) phosphoenzyme intermediates exist on the reaction path of alkaline phosphatase of Escherichia coli. Zn(II) and Cd(II) alkaline phosphatases both form E-P and E X P from inorganic phosphate. These intermediates show well separated 31P NMR resonances in slow chemical exchange with respect to each other and to unbound phosphate. The 31P signals of E X P of all forms of the 113Cd(II) enzyme are doublets (J = approximately 30 Hz) due to 113Cd-O-31P coupling. Heteronuclear decoupling shows the phosphate of E X P to be coordinated to the A site metal of the two metal ions, A and B, approximately 3.9 A apart at each catalytic center. The chemical shifts of E X P vary from approximately 4 ppm for the Zn(II) enzyme to 12.6-13.4 ppm for forms of the Cd(II) enzyme and indicate a major influence of the metal ions on the conformation around phosphorus. The phosphoryl group of E-P is not coordinated to either of the two metal ions at the active center as shown by the absence of 113Cd-O-31P coupling on the 31P signals of E-P formed by the 113Cd(II) enzymes. The chemical shift of E-P is not sensitive to metal ion species or stoichiometry and is 8-9 ppm for all forms of the Zn(II) and Cd(II) enzymes. The E-P in equilibrium E X P in equilibrium E + Pi equilibria are described by analogous pH functions for the Zn(II) and Cd(II) enzymes. At acid pH E-P predominates and is converted to E X P as the pH is raised, following a sigmoid pH profile. For the Zn(II) enzyme the midpoint of the E-P in equilibrium E X P equilibrium occurs at pH 5, while for the Cd(II)6 and Cd(II)2 enzymes the midpoints are pH 8.7 and 10, respectively. The ionization controlling the equilibrium between E-P and E X P may be that of a metal-bound H2O (-OH nucleophile) whose pKa will depend strongly on the hardness of the coordinating metal ion. For the Zn(II)4 enzyme one of 2 mol of E-P formed by the enzyme at acid pH dissociates readily at pH 7.5-8 where dissociation of E X P (Kd approximately equal to mM) is rate-limiting. Phosphate binds more tightly to the Cd(II) enzyme and 2 mol of phosphate remain bound until above pH 9 where E X P begins to dissociate at mM concentrations. The low Kd for E X P and the alkaline shift in the E-P in equilibrium E X P pH profile probably account for the slow turnover of the Cd(II) enzyme. Precise chemical shifts of the 113Cd and 31P NMR signals as well as the ratio of E-P/E X P at one active center of the dimer are altered by metal ion binding at the other active center indicating significant subunit-subunit interactions.