The Fe(III)Zn(II) form of recombinant human purple acid phosphatase is not activated by proteolysis.
The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe(3+)Fe(2+) human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in pK(es,1) (the value of an acid dissociation constant of the ES complex). These differences are due to the presence or absence, respectively, of an interaction between an aspartate residue in an exposed loop of the protein and one or more active site residues. To further explore the origin of these differences, the ferrous ion of recHPAP has been replaced by zinc. Analysis of the reconstituted Fe(3+)Zn(2+)recHPAP reveals an unexpected catalytic activity versus pH profile, in that the optimal pH is 6.3, similar to that of the proteolytically cleaved form (6.5). Moreover, replacement of the ferrous ion by zinc increases the turnover number more than 10-fold; the pK(es) values are also shifted as expected for the change in the divalent metal ion. Although the EPR spectra of both single polypeptide and proteolytically cleaved Fe(3+)Zn(2+)-recHPAP are independent of pH over the range 4.5-6.2, the visible spectrum of Fe(3+)Zn(2+)-recHPAP is pH dependent. These results suggest that the properties and environment of the divalent metal are important in determining the catalytic properties of mammalian PAPs, and in particular that a solvent molecule coordinated to the divalent metal ion may play a critical role in the catalytic cycle of these enzymes.
Funhoff
Enrico G
EG
SILS, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
Bollen
Mirko
M
Averill
Bruce A
BA
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D010710:Phosphates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin; D015032:Zinc / Q000737:chemistry
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D010710:Phosphates; D011994:Recombinant Proteins; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D014357:Trypsin; D015032:Zinc; D005459:Fluorides