The binding of cobalt(II) to apo alkaline phosphatase

Abstract

Alkaline phosphatase from Escherichia Coli is a dimeric zinc enzyme which catalyzes the hydrolysis of monophosphoric esters; it contains four zinc(II) and two magnesium(II) ions per molecule. Each subunit contains a ‘catalytic’ zinc ion, a ‘structural’ zinc ion and a ‘regulatory’ magnesium ion. The low resolution crystal structure shows that the catalytic and structural zinc ions are about 5 Å apart, while the magnesium ion is about 30 Å away from the above-mentioned ions [1]. The enzyme can be totally depleted of metal ions and reconstituted by the addition of two, four or six like or unlike metal ions. Since a high resolution X-ray structure is not yet available, the geometry of the metal sites and the donor group should be argued only from different spectroscopic data. Otvos et al. proposed, on the basis of 113Cd and 13C NMR data, that the catalytic metal ion could be coordinated by four histidine nitrogens [2]. Previous water proton NMR data indicated that a water molecule is present in the first coordination sphere of Mn 2+, Cu 2+ and Co 2+ alkaline phosphatase, substituted st the catalytic sites [3]. Combining these data, the catalytic metal ion should be five-coordinated; this hypothesis is in agreement with our recent proposal of five coordination of cobalt(II) ion in the catalytic site, based on the relatively low molar absorbance of the electronic spectra [4]. Structural and regulatory sites were assigned as pseudo-octahedral on the basis of the low molar absorbance of the cobalt(II) derivative. We have titrated apoalkaline phosphatase solutions at different pH values with increasing amounts of cobalt(II) ions in order to shed light on the distribution of metal ions among the various metal sites, which is still a matter of discussion. We worked with unbuffered and unsalted solutions either in the presence or absence of magnesium(II) ions. When excess Mg 2+ is present, only two cobalt(II) ions are required to develop fully the electronic spectrum typical of the catalytic site, while in its absence four cobalt(II) ions are required. We thus propose that when magnesium is present, cobalt(II) has a higher affinity for the catalytic site than for the structural sites, while in the absence of magnesium the affinity of cobalt(II) ions for the two sites is comparable; this holds both for the low and high pH limits. We also recorded the spectrum of cobalt(II) in the structural site at low pH in the presence of a 2:1 ratio of copper(II) ions to apoenzyme molecule, where copper is selectively bound only to the structural sites [5]; the difference spectrum gives a molar absorbance of around 10 per cobalt, which we assign to the cobalt(II) ions bound in the pseudo-octahedral environment of the structural site. It was previously reported that for the system M 2AP only one mol of inorganic phosphate is required to obtain to limit spectrum for the phosphate adduct [6], while for the M 4AP system, two equivalents of phosphate are required. We have titrated both Co 2Mg 4AP and CO 4Mg 2AP with inorganic phosphate and we found that in both cases only one mol of phosphate is required to develop fully the spectrum of the phosphate adduct. This result is in contrast with the model worked out for the enzymatic activity of AP [7].