title: Metal-ion mutagenesis: conversion of a purple acid phosphatase from sweet potato to a neutral phosphatase with the formation of an unprecedented catalytically competent Mn(II)Mn(II) active site.
abstract: The currently accepted paradigm is that the purple acid phosphatases (PAPs) require a heterovalent, dinuclear metal-ion center for catalysis. It is believed that this is an essential feature for these enzymes in order for them to operate under acidic conditions. A PAP from sweet potato is unusual in that it appears to have a specific requirement for manganese, forming a unique Fe(III)-mu-(O)-Mn(II) center under catalytically optimal conditions (Schenk et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 273). Herein, we demonstrate, with detailed electron paramagnetic resonance (EPR) spectroscopic and kinetic studies, that in this enzyme the chromophoric Fe(III) can be replaced by Mn(II), forming a catalytically active, unprecedented antiferromagnetically coupled homodivalent Mn(II)-mu-(H)OH-mu-carboxylato-Mn(II) center in a PAP. However, although the enzyme is still active, it no longer functions as an acid phosphatase, having optimal activity at neutral pH. Thus, PAPs may have evolved from distantly related divalent dinuclear metallohydrolases that operate under pH neutral conditions by stabilization of a trivalent-divalent metal-ion core. The present Mn(II)-Mn(II) system models these distant relatives, and the results herein make a significant contribution to our understanding of the role of the chromophoric metal ion as an activator of the nucleophile. In addition, the detailed analysis of strain broadened EPR spectra from exchange-coupled dinuclear Mn(II)-Mn(II) centers described herein provides the basis for the full interpretation of the EPR spectra from other dinuclear Mn metalloenzymes.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Queensland, Australia, 4072.
authors.forename: Natasa
authors.initials: N
authors.lastname: Mitić
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Noble
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gahan
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Hanson
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027723:Ipomoea batatas / Q000201:enzymology*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D008345:Manganese; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases