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.
Key indicatorsSingle-crystal X-ray study T = 293 K Mean '(C±C) = 0.004 A Ê R factor = 0.025 wR factor = 0.071 Data-to-parameter ratio = 13.3For details of how these key indicators were automatically derived from the article, see http://journals.iucr.org/e. # 2003 International Union of Crystallography Printed in Great Britain ± all rights reservedIn the mononuclear title complex, [Zn(PMG) 2 ]Á2H 2 O or [Zn(C 8 H 9 N 2 O 2 ) 2 ]Á2H 2 O, the Zn II center is surrounded by two N-(2-pyridylmethyl)glycinate (PMG) ligands, which impose a distorted octahedral environment on the metal. Two deprotonated molecules of the new tridentate N,N H ,O-donor ligand HPMG are facially coordinated to the Zn II center in such a way that the atoms of the same kind are mutually trans to each other, generating a centrosymmetric structure.
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