The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

Abstract

The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe(3+)-Fe(2+) pig PAP (uteroferrin; Uf) and its Fe(3+)-Mn(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe(2+) by Mn(2+) lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a mu-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe(3+) site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe(3+). The faster rates of interaction with the Fe(3+) site in the Fe(3+)-Mn(2+) derivative than the native Fe(3+)-Fe(2+) form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.

Figure 1

Proposed nucleophilic hydroxide molecules for PAP-catalyzed hydrolysis are positioned either: (i) terminal to Fe³⁺; (ii) bridging the two metal ions; or (iii) residing in the second coordination sphere.

Figure 2

Top panel: pH dependence of k_cat (s⁻¹) for Fe³⁺-Fe²⁺ (filled circles) and Fe³⁺-Mn²⁺ (open circles) Uf. Bottom panel: pH dependence of k_cat/K_m (s⁻¹ mM⁻¹) for Fe³⁺-Fe²⁺ (filled circles) and Fe³⁺-Mn²⁺ (open circles) Uf. Data were measured using pNPP as substrate.

Figure 3

First-order rate constants (k_obs; s⁻¹) for FeFe Uf (red) and FeMn Uf (blue) at pH 4.9, 25°C, I= 0.1 M (NaCl). Substrate concentrations ranged from 3.5 mM to 50mM. Data for P_i were reproduced from reference .

Figure 4

Depiction of a mechanism for PAP-catalyzed substrate hydrolysis consistent with the stopped-flow kinetic data. K_ass1 and K_ass2 represent formation of first a non-competent complex, and then an ES complex in which substrate coordinates to the divalent metal ion. Binding of a second, inhibitory substrate molecule occurs with association constant K_I. Hydrolysis (k_cat) occurs from attack by a second-sphere nucleophilic water (see text for additional discussion). Available crystal structures show the presence of several such waters in the outer coordination sphere of the metal center. The nucleophilic water is shown in ES’ in the lower part of the figure, but omitted from the upper structures for clarity.

Scheme 1

Scheme 2

Schematic representation of the nucleophilic attack by a second coordination sphere water molecule on the phosphorus atom of the substrate.