Multiple binding sites for tetrahedral oxyanion inhibitors of bovine spleen purple acid phosphatase

Abstract

The theory of multiple inhibition kinetics has been extended to enzymes for which one inhibitor is noncompetitive and the other exhibits mixed inhibition. Plots of reciprocal velocity versus the concentration of either inhibitor at various fixed concentrations of the second inhibitor are predicted to give parallel lines if binding of the inhibitors is mutually exclusive and intersecting lines if the inhibitors interact at different sites on the enzyme. Application of this analysis to the purple acid phosphatase from bovine spleen in the presence of molybdate (a noncompetitive inhibitor) and phosphate (which exhibits mixed inhibition) results in parallel lines in the reciprocal velocity plots, indicating that phosphate and molybdate compete for a common site; since molybdate is a noncompetitive inhibitor, this site is inferred to be distinct from the site at which substrate binds and is hydrolyzed. Extension of these ideas suggests that phosphate ester substrates should be capable of binding to the molybdate-binding site as well as to the active site, and evidence for substrate inhibition at high substrate concentrations has been obtained. The implications of these findings for interpretation of previous spectroscopic studies of purple acid phosphatase complexes with tetrahedral oxyanions are discussed.

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

“…Conversely, many plant APs, including tomato SAP2, are competitively inhibited by P i [1]. The different modes of P i inhibition may arise from differences in structural and/or conformational properties of APs [35]. Inhibition by P i is in accord with our hypothesis that tomato IAP plays an important P i -recycling role during P i -starvation, when cellular P i levels may be decreased by up to 50-fold [5].…”

Structural, Spectroscopic, and Theoretical Investigation of a T-Shaped [Fe3(μ3-O)] Cluster

“…Consequently, the quest for high-spin iron­(III) clusters that exhibit unusual magnetic properties or function as enzymatic model complexes is an active area of research and, to date, has led to the characterization of numerous polynuclear ferric complexes with interesting chemistry, spin topologies, magnetic, and electronic structures . In biological systems, diferric clusters are abundant and often encountered in the resting state of nonheme enzymes, such as the oxygen transport protein hemerythrin, purple acid phosphatase, ribonucleotide reductase, soluble methane monooxygenase, and the ferroxidation site of ferritins . In contrast, trinuclear iron­(III) clusters are considerably less common with only a handful of examples having been identified so far, such as the iron–sulfur clusters of ferredoxins and aconitase enzymes, the oxo-bridged cluster of ferreascidin, and the ferroxidase site of some bacterial ferritins. , …”

A new di-m-sulfate complex as a model of purple acid phosphatase-sulfate complexes

“…The interaction of the active site of purple acid phosphatases (PAPs) with sulfate and other perturbants has been described 1,2 . This oxyanion is known to be able to interact with the reduced enzymes, which in the presence of air form the oxidized PAPox-sulfate complexes.…”