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Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and⁄or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypep-tide PAP. | iFEBS Journal Substrate positioning by His92 is important in catalysis by purple acid phosphatase Enrico G. Funhoff1 4 Yunling Wang2 Goran Andersson2 and Bruce A. Averill1 3 1 Swammerdam Institute for Life Sciences University of Amsterdam the Netherlands 2 Karolinska Institutet Division of Pathology Huddinge University Hospital Sweden 3 Department of Chemistry University of Toledo OH USA 4 Institute of Biotechnology HPT ETH Honggerberg Zurich Switzerland Keywords kinetics mechanism mutagenesis purple acid phosphatase spectroscopy Correspondence B. A. Averill Department of Chemistry University of Toledo 2801 West Bancroft Road Toledo Ohio 43606-3390 USA Fax 1 419 5301586 Tel 1 419 5301585 E-mail baa@utoledo.edu Website http www.chem.utoledo.edu FAC_INFO Bruce SOURCE.htm Received 12 January 2005 revised 13 March 2005 accepted 28 March 2005 doi 10.1111 j.1742-4658.2005.04686.x Proteolysis of single polypeptide mammalian purple acid phosphatases PAPs results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala which eliminates polar interactions of this residue with the active site resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely conversion of this residue into Asn which cannot function as either a proton donor or acceptor but can provide hydrogen-bonding interactions resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima with pK Cs_ values consistent with the involvement of an iron III hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results together with EPR data support a role of His92 in positioning either the .
