Manganese(II) as a Probe of the Active Center of Alkaline Phosphatase

Abstract

Alkaline phosphatase (AP) contains three metal ion binding sites at each active center, sites A, B, and C, forming a triangle of sides 3.9, 4.9, and 7.0 A. When two Mn(II) ions occupy the two A sites on the AP dimer, ESR spectra at 9.1 and 35 GHz show the A sites to have both axial and rhombic distortions from cubic symmetry (D = 0.083 cm-1, E = 0.013 cm'1). Phosphate binding to the A site causes small but detectable changes in symmetry around the Mn(II) (D = 0.070 cm-1, E = 0.017 cm-1). Mn(II) in the B sites shows an ESR spectrum suggesting a regular geometry. Water 'H Tf]values of solutions of apoalkaline phosphatase titrated with 1–4 Mn(II) ions/dimer show that the Mn(II) ions in the A sites carry exchanging water molecules (probably 2), while water molecules bound to the B site, if present, are in slow exchange, since the B-site Mn(II) makes little contribution to the relaxivity of the enzyme. NMRD profiles from 0.01 to 50 MHz of Mn2E2AP (E for “empty”), Mn2Mg2AP, and Mn2Mn2AP show the coordinated water in the A sites to be in relatively slow exchange; i.e., rM£ 10–6s. Phosphate binding reduces the relaxivity of all forms of the Mn(II) enzyme by approximately 50%. The phosphate ligand appears to displace one of two H20 molecules originally present at the A site. Thus, the A-site Mn(II) appears to be 5-coordinate with three His and two H20 ligands. While turnover of the Mn(II) enzymes is 0.3% of that of the Zn(II) enzyme, all the phosphoenzyme intermediates form and the water structure of the Mn(II) enzyme is probably similar to that of the Zn(II) enzyme. © 1989, American Chemical Society. All rights reserved.