Copper(II) as a Probe of the Active Centers of Alkaline Phosphatase

Abstract

Alkaline phosphatase (AP) contains three metal ion binding sites at each active center A, B, and C, forming a triangle of 3.9, 4.9, and 7 A between the three metal ions. WaterlH T1-lof solutions of metal-free apoalkaline phosphatase and of derivatives containing 1, 2, 3, and 4 mol of Cu(II)/mol of enzyme dimer have been measured between 0.01 and 40 MHz (NMRD). Two Cu(II) ions added to the apoenzyme sequentially occupy the A sites at both pH 6 and pH 8, giving rise to NMRD typical of Cu(II) coordinated to exchangeable water molecules and bound to isolated sites in macromolecules. A d-d absorption maximum at 690 nm (∊/Cu(II) = 145 M-1cm-1) and an axially symmetric ESR signal (g∥= 2.32, g⊥= 2.09, A∥164 X 10–4cm-1) show the Cu(II) ions in the Cu2AP to occupy identical sites. The third and fourth equivalents of Cu(II) added to the enzyme result in the quenching of the relaxivity of the Cu2E2AP (E for “empty”) as well as a loss of the room-temperature ESR signal. Magnetic susceptibility measurements (room temperature) show that there is strong antiferromagnetic coupling between the Cu(II) ions in the A and B sites (J ⋍ 120 cm-1). Part of the loss of the relaxivity on the binding of the second pair of Cu(II) ions to alkaline phosphatase can be attributed to the magnetic interaction between the Cu(II) ions at sites A and B. This interaction may reflect the presence of a bridging ligand between CuA and CuB, either OH-or Ser-O-.31P NMR shows that phosphate binds to only one monomer of Cu2E2AP; i.e., phosphate binding is negatively cooperative. Phosphate binding is accompanied by a time-dependent shift in the d-d transitions to give two absorption maxima, one at the original 690 nm and a new one at 790 nm. Simultaneously, the room-temperature ESR spectrum decreases in amplitude and the paramagnetic component of the water proton relaxation rate decreases. These findings suggest that phosphate binding to Cu2E2AP induces the migration of Cu(II) from the A site of the phosphate-free monomer to the B site of the phospho monomer. These data are analyzed in terms of the water and ligand structure at the active centers of the Cu(II) derivatives of alkaline phosphatase. © 1989, American Chemical Society. All rights reserved.