Abstract
Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (Ki = 40 µmol L-1) while it inhibits red kidney bean PAP competitively (Ki = 30 µmol L-1). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.
Highlights
At that pH vanadate monomers tend to oligomerise in solution provided the total concentration of the tetraoxo anion is ≥ 0.25 mmol L-1.23 Since the monomeric form is the main inhibitor of PAPs23 it was essential to estimate the concentration of this species under conditions employed in the kinetic assays
In contrast to the red kidney bean enzyme, for pig Purple acid phosphatases (PAPs) the inhibition mode appears to be non-competitive with Ki = Kic = Kiuc
Based on the similarity of the inhibition data it appears likely that red kidney bean PAP has a transition state similar to that observed in E. coli alkaline phosphatase
Summary
Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases, with members differing widely with respect to physicochemical properties, tissue localisation and proposed biological function.[1,2] The characteristic purple colour of these enzymes is due to a charge transfer transition between a ligand (tyrosinate) donor orbital and an acceptor orbital on the ferric ion.[1,3,4] PAPs catalyse the hydrolysis of a variety of phosphorylatedPlant and mammalian PAPs have very similar geometries at the catalytically relevant active site with all seven metal ion-coordinating residues being invariant.[1,6,13,14,15,16,17,18] Additional residues that play important roles in various aspects of catalysis are wellVol 17, No 8, 2006 conserved, including the proposed proton donor to the leaving group (His[296] and His[195] in red kidney bean and pig PAP, respectively).[13,14,15,16,17,18] The observations that PAPs from different organisms have similar Km values for a variety of substrates and are inhibited to a similar extent by inorganic phosphate illustrate the structural similarity of the active sites of different PAPs.[10,11,19,20,21]The inhibition of reactivity by a range of antagonists is a common method to investigate an enzyme’s catalytic mechanism; such a study may provide valuable information and guidance for the design and development of specific and potent drugs. The inhibitory effects of fluoride and vanadate on the activity of red kidney bean and pig PAP are assessed and compared with those measured for other PAPs. The mechanistic implications of these studies are discussed.
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