title: 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 kcat 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 mu-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.
authors.affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, QLD, 4072, Australia. schenk@uq.edu.au
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
authors.forename: Rosely A
authors.initials: RA
authors.lastname: Peralta
authors.forename: Suzana Cimara
authors.initials: SC
authors.lastname: Batista
authors.forename: Adailton J
authors.initials: AJ
authors.lastname: Bortoluzzi
authors.forename: Bruno
authors.initials: B
authors.lastname: Szpoganicz
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Dick
authors.forename: Paul
authors.initials: P
authors.lastname: Herrald
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Hanson
authors.forename: Robert K
authors.initials: RK
authors.lastname: Szilagyi
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Riley
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gahan
authors.forename: Ademir
authors.initials: A
authors.lastname: Neves
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D002413:Cations, Divalent; D007527:Isoenzymes; D008670:Metals; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase