Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR.

P Gettins,J E Coleman

doi:10.1016/s0021-9258(18)90618-6

Abstract

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.

Highlights

Chloride binding to alkaline phosphatase fErosmch- relatively specific binding sitefor halide ions, the intensityof erichia coli has been monitored by 35ClNMR for the native zinc enzyme andby '13Cd NMR for twoCd(I1)substitutedspecies,phosphorylatedCd(II)6alkaline phosphataseandunphosphorylatedCd(II)2alkaline phosphatase
Shift changes produced in the A site '13Cd resonance There have been severalpreviously published papers aimed for the covalent (E-P)form of the enzyme uersus the at studying C1- interactions with alkaline phosphataseby 35Cl noncovalent ( E .P) form of the enzyme, it is concluded NMR, looking at the broadening of the bulk C1- resonance that theA site metal ion can assume a five-coordinate form
Specific broadening of the 35Clresonance from bulCk1- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggestingan influence by occupancy of the B site on the interactioonf monodentate ligands at theA site

Summary

Introduction

An earlierreport from thislaboratoryhas clearly demonstratedthegreatsensitivity of the chemical shift of 1'3Cd(II)occupying the A site of Cd(II)2alkaline polar broadening is observed upon phosphate binding phosphatase to variationsin C1- concentration [9]. Theadvantage of this species over other unphosphorylated Cd(II), alkaline phosphatase or Cd(II), alkaline phosphatase is that chemical exchangemodulation in theunphosphorylated forms of the latter produceextremelybroad resonances[12].Suchan asymmetrically occupied enzyme species was prepared by addition of 2 equivalents of cadmium-113 acetate to apoenzyme at pH 6.5 in the presence of 0.1 M NaCl. Under these conditions, thecadmium occupies both A sites and results in a cadmium NMR spectrum of one resonance at 169 ppm.This was checked for the presentsample.

Results

Conclusion