Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Abstract

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k cat of the Fe(III)Ni(II) derivative (approximately 60 s -1 ) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the l-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency.

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

“…Phosphatase activities of the Fe(III)-M(II) (M is Mn, Co, Ni, Cu or Zn) complexes of BPBPMP 2-have been measured with the activated substrate 2,4-bis(dinitrophenyl) phosphate (BDNPP) [9][10][11]. These reactions are strongly dependent on pH, giving rise to bell-shaped pH versus rate profiles and pH optima in the range 6.0-7.0 [10][11][12][13][14]. In each case the catalytically active species is proposed to be of the type [(OH)Fe(III)(l-OH)M(II)(OH 2 )] [1].…”

GaIII Complexes as Models for the MIII Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate

“…In model (b) the bridging (hydr)oxo group acts as nucleophile. 5,10,12,13,14 The catalytic activity of a metal ion is largely governed by its Lewis acidity. The stronger the Lewis activity, the stronger is the effect of the metal ion on substrate activation.…”

Metal-Ion Mutagenesis: Conversion of a Purple Acid Phosphatase from Sweet Potato to a Neutral Phosphatase with the Formation of an Unprecedented Catalytically Competent MnIIMnII Active Site

“…16,17 In fact, this approach has been successfully employed to generate metalloderivatives of PAPs. [16][17][18][19][20][21][22][23] For instance, the Fe II in pig PAP (uteroferrin) can be replaced by Zn II without a significant change in activity, whereas the Mn II , Ni II , Cu II , and Co II derivatives display 10-50% activity of the native enzyme. 19,[21][22][23] Although the divalent ion can be replaced, there are no reports of catalytically active dinuclear homodivalent PAPs.…”