Substrate positioning by His92 is important in catalysis by purple acid phosphatase

Abstract

The binuclear metalloenzyme purple acid phosphatase (PAP) [1], which may be involved in disorders such as osteoporosis [2][3][4], Gaucher disease [5], hairy cell leukemia [6], and AIDS [7] is widely distributed in mammalian tissues [8,9]. The expression level of PAP [also referred to as tartrate resistant acid phosphatase (TRAP) or type 5 acid phosphatase (AcP5; EC 3.1.3.2)] is elevated in these disorders, suggesting a relationship between the increased levels of the enzyme and the clinical picture. The presumed role of PAP makes it important to develop drugs that can inhibit PAP activity. In order to facilitate this process, the precise catalytic mechanism must be elucidated, including the function of each residue involved in catalysis.Although the available crystal structures of PAPs [10][11][12] provide structural information about the residues potentially involved in catalysis, to date only structures of inactive redox and protonation states of the enzymes have been reported. All structures show an active site composed of two metal ions bridged by a solvent-derived species and a bidentate aspartate residue. In addition, the iron(III) ion is coordinated by a tyrosinate [resulting in a ligand-to-metal charge transfer (LMCT) transition that is responsible for the 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 es,1 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 nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.Abbreviations kPP, protein phosphatase from phage k; KBPAP, kidney bean PAP; LMCT, ligand-to-metal charge transfer; MOI, multiplicity of infection; PAP, purple acid phosphatase; p-NPP, para-nitrophenylphosphate; PP, protein phosphatase; recHPAP, recombinant human purple acid phosphatase; recRPAP, recombinant rat PAP; TRAP, tartrate resistant acid phosphatase.

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

“…In agreement with previous studies [40,[45][46][47] pK es2 is assigned to a conserved histidine residue in the second coordination sphere, which is likely to act as a proton donor to the leaving alcohol group during catalysis [14,[47][48][49]. Site-directed mutagenesis studies identified His92 as the likely residue [47].…”

“…In comparison with the native Fe(III)Fe(II) form of Uf each pK a in the Fe(III)Ni(II) derivative is shifted towards more alkaline values, resulting in a corresponding shift of the pH for optimum reactivity from 5.0 to 5.5 upon replacing Fe(II) with Ni(II). Based on an inspection of the active site of the enzyme and previous studies [40,46,47], the following assignment is proposed:…”

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

“…21 For metallohydrolases, fluoride and vanadate are known inhibitors which have been shown to coordinate directly to the metal ion(s) in the active site and thus interfere with catalytic turnover. 1,20,[23][24][25][26][27][28] The two inhibitors are likely to affect different aspects of catalysis and hence may provide insight into different steps of the reaction mechanism.…”

“…21 For metallohydrolases, fluoride and vanadate are known inhibitors which have been shown to coordinate directly to the metal ion(s) in the active site and thus interfere with catalytic turnover. 1,20,[23][24][25][26][27][28] The two inhibitors are likely to affect different aspects of catalysis and hence may provide insight into different steps of the reaction mechanism.The ability of fluoride to readily replace nucleophilic hydroxide groups can provide (i) an insight into the number of labile water/hydroxide binding sites in the active site, and (ii) the role of the terminally bound or bridging hydroxides in the reaction mechanism. 26,29 Fluoride, a well established osteogenic agent which promotes osteoblastic proliferation and differentiation at clinically relevant concentrations, inhibits animal PAPs in a pH-dependent manner.…”

Identification of Aspartic Acid and Histidine Residues Mediating the Reaction Mechanism and the Substrate Specificity of the Human UDP-glucuronosyltransferases 1A

“…Thus, it will be important to take into account the role of this key residue in further development of predictive models of the specificity of UGT1A4, UGT1A3, and UGT1A1. Similarly, Funhoff et al (40) showed recently that a histidine residue plays an important role in positioning the substrate of the purple acid phosphatase, and Watts et al (41) identified a single active site histidine residue governing substrate selection of aromatic lyase family.…”