New insights into the mechanism of purple acid phosphatase through (1)H NMR spectroscopy of the recombinant human enzyme.
Proton NMR spectra of FeIII-FeII recombinant single polypeptide human PAP (recHPAP) have been measured at, above, and below its pH optimum, as have the spectra of inhibited forms containing fluoride and phosphate, analogues of the substrates hydroxide and phosphate esters, respectively. The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to the dinuclear center. Thus, the group that is responsible for pKa,1 in the enzymatic activity versus pH profile functions as a "gatekeeper", whose protonation state controls anion binding to the mixed-valent dinuclear site. The correlation between the pKa values observed in kinetics studies and for the spectroscopic changes strongly suggests that this group is the nucleophilic hydroxide that attacks the phosphate ester substrate.
Dikiy
Alexander
A
Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, I-40127 Bologna, Italy.
Funhoff
Enrico G
EG
Averill
Bruce A
BA
Ciurli
Stefano
S
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D006023:Glycoproteins; D011522:Protons; D011994:Recombinant Proteins; C091137:purple acid phosphatase; D000135:Acid Phosphatase