Inhibition of Bacteriophage λ Protein Phosphatase by Organic and Oxoanion Inhibitors†

Bacteriophage λ protein phosphatase (λPP) with Mn2+ as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of λPP, albeit with inhibition constants (Ki) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (Ki ) 5.1 ( 1.6 μM) whereas phosphonoacetic acid (Ki ) 380 ( 45 μM) and acetohydroxamic acid (Ki > 75 mM) modestly inhibited λPP. Low-temperature EPR spectra of Mn2+-reconstituted λPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn2+ ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn)2 dimer is most prominent upon the addition of orthovanadate (Ki ) 0.70 ( 0.20 μM) and PhAH, indicating that these inhibitors tightly interact with the (Mn)2 form of λPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by λPP and other serine/threonine protein phosphatases. Eukaryotic serine/threonine phosphoprotein phosphatases utilize a dinuclear metal cluster for catalysis and are involved in the dephosphorylation of a variety of phosphoserine/ threonine substrates (3-7). These enzymes function in crucial cellular processes, including gene expression, cell growth, and cell differentiation. Numerous pathophysiological conditions have been attributed to aberrant regulation of these phosphatases including apoptosis, cardiac hypertrophy, immunosupression, memory loss, and cancer (8-11). Serine/threonine phosphoprotein phosphatases fall into two structurally distinct gene families (PPP1 and PPM) according to nomenclature adopted for human genes (12). These phosphatases have also been classified on the basis of their substrate specificity, divalent metal ion dependence, and inhibition by different phosphatase inhibitors (13-16). Of the enzymes in the PPP family, protein phosphatases 1 (PP1), 2A (PP2A), and calcineurin (PP2B) contain the phosphoesterase sequence motif, DXH(X)nGDXXDG(X)mGNHD/E (5, 17, 18). X-ray structures of PP1 (3, 4), calcineurin (2, 19), and, most recently, bacteriophage λ protein phosphatase (λPP) (1), a related serine/threonine phosphoprotein phosphatase of the PPP family, reveal that this phosphoesterase motif forms a â-R-â-R-â secondary structure scaffold that provides ligands for the active site dinuclear metal cofactor. An identical constellation of metal ligands and coordination geometries is found in all three enzymes, with the two metal ions linked by a bridging solvent molecule and a μ-1,1 aspartic acid. Additional metal ligands include two solvent molecules, a histidine, and an aspartic acid to the first metal ion (denoted the M1 metal site) and one solvent molecule, two histidines, and an asparagine to the second metal ion (denoted M2). The active sites of these three enzymes are nearly superimposable, with root-meansquare deviation between active site metals and protein ligands <0.5 Å.2 The conservation in amino acid sequence, protein fold, and active site structure suggests that phos† This work was supported by Grant GM46865 from the National Institutes of Health. * To whom correspondence should be addressed at the Mayo Clinic and Foundation, 200 First St. S.W., Rochester, MN 55905. Telephone: (507) 284-4743. Fax: (507) 266-9302. E-mail:[email protected]. 1 Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; PhAH, phosphonoacetohydroxamic acid; λPP, bacteriophage λ phosphoprotein phosphatase; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, calcineurin (protein phosphatase 2B); PPM, eukaryotic Ser/Thr phosphatase subfamily including PP2C; PPP, eukaryotic Ser/Thr phosphatase subfamily including PP1, PP2A, and calcineurin; pNPP, p-nitrophenyl phosphate; PTP, protein tyrosine phosphatase family; WT, wild type. 2 The root-mean-square deviation calculation was performed using the SwissPdbViewer (version 3.5) software program. The atomic coordinates of λPP, calcineurin, and PP1 were retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein DataBank (http://www.rcsb.org/pdb/) under filenames 1G5B, 1AUI, and 1FJM, respectively. Active site metal ions and amino acid residues D20, H22, D49, N75, H139, H186, and H76 of the A, B, and C subunits of crystallized λPP (1) were superimposed with active site metal ions and amino acid residues D90, H92, D118, N150, H199, H281, and H151 of the calcineurin A subunit (2) and the active site metal ions and amino acid residues D64, H66, D92, N124, H173, H248, and H125 of PP1 (3). 1051 Biochemistry 2002, 41, 1051-1059 10.1021/bi011577b CCC: $22.00 © 2002 American Chemical Society Published on Web 12/21/2001 phomonoester hydrolysis proceeds through a common mechanism in these three enzymes. Purple acid phosphatases can also be considered to be members of the PPP family of metallophosphoesterase. However, the phosphoesterase motif of purple acid phosphatases, D(X)nGDXXY(X)mGNHD/E, represents a variation of the motif noted above (16). These similarities are reflected in a comparable but not identical active site geometry compared to calcineurin and PP1. In PP1/calcineurin, a histidine ligand to the Fe ion is replaced by a tyrosine residue in the purple acid phosphatases. An additional substitution of a solvent molecule with a histidine ligand results in a net water-for-tyrosine substitution in the Fe coordination sphere of calcineurin/PP1 compared to purple acid phosphatase (10). Mn2+ is proposed to be the physiological metal ion cofactor for λPP3 and is an excellent metal coactivator (20). In addition, it has been proven to be a useful active site probe because of its paramagnetic properties. Characterization of the spin-coupled (Mn)2 center of λPP by electron paramagnetic resonance (EPR) spectrometry has demonstrated that the M1 and M2 metal binding sites exhibit different binding affinities, with the M2 site as the high-affinity site (21, 22). These results, along with the recently solved X-ray crystal structure and the biochemical and spectroscopic analysis of proteins in which active site residues were mutated (1, 22, 23), have provided considerable mechanistic and structural information. Taken together, these studies confirm λPP as an excellent model of the more complex, eukaryotic Ser/Thr PPases. A study of the interaction of serine/threonine phosphoprotein phosphatases with various inhibitors is important as it may provide clues to the mechanism of phosphate ester hydrolysis (24, 25). It is long established that one of the products of the phosphatase reaction, orthophosphate, is a modest inhibitor of PPPs (Ki ∼ 1 mM) at physiological pH (20, 26, 27). Other structural and electronic oxoanion analogues of orthophosphate, such as orthovanadate, molybdate, sulfate, tungstate, and arsenate, also inhibit PPPs and purple acid phosphatases (20, 26, 28-