Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A

Botulinum neurotoxin serotype A (BoNTA) is one of the most toxic substances known. Currently, there is no antidote to BoNTA. Small molecules identified from high-throughput screening reportedly inhibit the endopeptidase--the zinc-bound, catalytic domain of BoNTA--at a drug concentration of 20 microM. However, optimization of these inhibitors is hampered by challenges including the computational evaluation of the ability of a zinc ligand to compete for coordination with nearby residues in the active site of BoNTA. No improved inhibitor of the endopeptidase has been reported. This article reports the development of a serotype-selective, small-molecule inhibitor of BoNTA with a K(i) of 12 microM. This inhibitor was designed to coordinate the zinc ion embedded in the active site of the enzyme for affinity and to interact with a species-specific residue in the active site for selectivity. It is the most potent small-molecule inhibitor of BoNTA reported to date. The results suggest that multiple molecular dynamics simulations using the cationic dummy atom approach are useful to structure-based design of zinc protease inhibitors.     

Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K _i ^app value of 3.8±0.8 µM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC₅₀ value of 100 µM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 Ų for BoNTAe is about four times larger than the typical protein-protein interface area of 750–1,500 Ų. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K _i value of 760±170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites.     

Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A

Botulinum neurotoxin serotype A (BoNTA) is highly toxic, and its antidote is currently unavailable. The essential light-chain subunit of BoNTA is a zinc endopeptidase that can be used as a target for developing antidotes. However, the development of high-affinity, small-molecule inhibitors of the endopeptidase is as challenging as the development of small-molecule inhibitors of protein-protein complexation. This is because the polypeptide substrate wraps around the circumference of the endopeptidase upon binding, thereby constituting an unusually large substrate-enzyme interface of 4840 angstroms2. To overcome the large-interface problem, we propose using the zinc-coordination and bivalence approaches to design inhibitors of BoNTA. Here we report the development of alkylene-linked bis-imidazoles that inhibit the endopeptidase in a two-site binding mode. The bis-imidazole tethered with 13 methylene groups, the most potent of the alkylene-linked dimers, showed 61% inhibition of the zinc endopeptidase of BoNTA at a concentration of 100 microM. The results demonstrate the presence of a peripheral binding site for an imidazolium group at the rim of the BoNTA active-site cleft. This peripheral site enables the use of the bivalence approach to improve our previously reported small-molecule inhibitors that were developed according to the zinc-coordination approach.     

Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations

Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by approximately 1 A in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.     

Inhibition of Na+/Ca2+ exchange in pituitary plasma membrane vesicles by analogues of amiloride

Amiloride is a weak inhibitor of Na+/Ca2+ exchange in isolated plasma membrane vesicles prepared from GH3 rat anterior pituitary cells. However, substitution on either a terminal guanidino nitrogen atom or the 5-amino nitrogen atom can increase inhibitory potency ca. 100-fold (I50 approximately 10 microM). A structure-activity study indicates that defined structural modifications of guanidino substituents are associated with increases in inhibitory activity. In contrast, analogues bearing 5-amino substituents generally increase in potency with increasing hydrophobicity of the substitution. Specificity in action of either class is indicated by several criteria. These inhibitors do not disrupt the osmotic integrity of the membrane, nor do they significantly interfere with plasmalemmal Ca2+-ATPase-driven Ca2+ uptake, Na+,K+-ATPase enzymatic activity, or the function of Ca2+ or K+ channels. Inhibition is freely reversible, further indicating a lack of nonspecific membrane effects. The mechanism by which each inhibitor class blocks exchange was found to be identical. Protonation of the guanidino moiety (i.e., cationic charge) is essential for activity. Analysis of transport inhibition as a function of Ca2+ concentration indicates noncompetitive kinetics. However, inhibition was reversed by elevating intravesicular Na+, indicating a competitive interaction with this ion. These results suggest that the inhibitors function as Na+ analogues, interact at a Na+ binding site on the carrier (presumably the site at which the third Na+ binds), and reversibly tie up the transporter in an inactive complex. In addition to blocking pituitary exchange, these analogues are effective inhibitors of the bovine brain and porcine cardiac transport systems.     

An investigation of bisphosphonate inhibition of a vacuolar proton-pumping pyrophosphatase

We report the results of a three-dimensional quantitative structure-activity relationship (3D-QSAR)/comparative molecular field analysis (CoMFA) of the activity of 18 bisphosphonates and imidodiphosphate in the inhibition of a mung bean (Vigna radiata L.) vacuolar proton pumping pyrophosphatase (V/H(+)-PPase; EC 3.6.1.1). We find an experimental versus QSAR predicted pK(app)(i) R(2) value of 0.89, a cross-validated R(2) = 0.77, and a bootstrapped R(2) = 0.89 for 18 bisphosphonates plus imidodiphosphate over the 1.3 microM to 425 microM range of K(app)(i) values. We also demonstrate that this approach has predictive utility (a 0.26 pK(app)(i) rms error for three test sets of 3 activity predictions each), corresponding to about a factor of two error in K(app)(i) prediction. The 3D-QSAR/CoMFA approach provides a quantitative method for predicting the activity of V/H(+)-PPase inhibitors and is likely to be of use in the design of novel pharmacological agents since all of the major human disease-causing parasitic protozoa contain large levels of pyrophosphate, together with V-type proton-pumping pyrophosphatases located in plant-like vacuoles (acidocalcisomes), which are absent in their mammalian hosts.     

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.     

Inhibition of choline oxidase by quinoid dyes

Choline oxidase catalyzes the oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. This study reports on the inhibitory effects of triarylmethanes (cationic malachite green; neutral leukomalachite green), phenoxazines (cationic, meldola blue and nile blue; neutral nile red) and a structurally-related phenothiazine (methylene blue) on choline oxidase, assayed at 25 degrees C in 50 mM MOPS buffer, pH 7, using choline as substrate. Methylene B acted as a competitive inhibitor with K(i) = 74 +/- 7.2 microM, pointing to the choline-binding site of the enzyme as a target site. Nile B caused noncompetitive inhibition of enzyme activity with K(i) = 20 +/- 4.5 microM. In contrast to methylene B and nile B, malachite G and meldola B caused complex, nonlinear inhibition of choline oxidase, with estimated K(i) values in the micromolar range. The difference in kinetic pattern was ascribed to the differential ability of the dyes to interact (and interfere) with the flavin cofactor, generating different perturbations in the steady-state balance of the catalytic process.     

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.     

Small Molecules Showing Significant Protection of Mice against Botulinum Neurotoxin Serotype A

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism that could afflict large, unprotected populations if the toxin were employed in an act of bioterrorism. Current post-exposure therapy is limited to symptomatic treatment or passive immunization that is effective for treating infant botulism at a cost of US $45,300 per treatment regimen. Antibodies can neutralize the extracellular but not the intracellular BoNTA. Moreover, antibody production, storage, and administration in a mass casualty scenario pose logistical challenges. Alternatively, small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are sought to antagonize the extracellular or intracellular toxin. While several such molecules reportedly demonstrated efficacy in protecting cells against BoNTA, there is scant information to show that small molecules can significantly protect mammals against BoNTA. Herein we report the development of effective small-molecules BoNTAe inhibitors with promising in vivo pharmacokinetics. One such molecule has an in vivo half-life of 6.5 hours and is devoid of obvious sign of toxicity. Pre-treatment with this molecule at 2 mg/kg protected 100% and 70% of treated mice against BoNTA at 5 times of its median-lethal dose during the periods of 2 and 4 half-lives of the inhibitor, respectively. In contrast, 40% and 0% of untreated mice survived during the respective periods. Similar levels of protection were also observed with two other small molecules. These results demonstrate that small molecules can significantly protect mice against BoNTA and support the pursuit of small-molecule antagonists as a cost-effective alternative or as an adjunct to passive immunity for treating botulism.     

Novel inhibitors of enkephalin-degrading enzymes. III: 4-Carboxymethylamino-4-oxo-3 (phenylamino) butanoic acids as enkephalinase inhibitors.

4-Carboxymethylamino-4-oxo-3-(4'-aminophenylamino) butanoic acid (25), its ethyl ester (26) and the corresponding unsubstituted-aryl analogues (17) and (16) are fairly potent inhibitors of enkephalinase (neutral endopeptidase; EC 3.4.24.11), Ki = 0.14-0.39 microM, with weak inhibitory potency, Ki = 15-75 microM, towards aminopeptidase MII. In the mouse abdominal constriction test, the esters (26) and (16) showed systemic inhibitory (antinociceptive) activity with ED50 values 62 +/- 3.05 and 81 +/- 1.74 mg/kg respectively. In the mouse tail immersion test, both (26) and (16) exhibited antinociceptive activity when administered intracerebroventricularly and (26) also exhibited a systemic effect which was only partially reversed by naltrexone. The antinociceptive effect seen with (26) reflects its ranking in vitro as an inhibitor of enkephalinase (Ki = 0.14 microM) but it is possible that this effect is not totally opioid-mediated. Compounds (26) and (16) represent the first combined inhibitors of enkephalinase and aminopeptidase MII.     

Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors

From L-alpha-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of substrate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised and coupled to a model pentapeptide furnishing a set of hexapeptide inhibitors. Promising inhibitory activities with K(i) values of 0.18 microM (11b, P1 electrophilic alpha,beta-unsaturated ketone), 0.46 microM (12e, P1 electrophilic alkyl ketone) and 0.98 microM (10e, P1 non-electrophilic alkenyl alcohol as diastereomeric mixture). The reference hexapeptide product inhibitor had a K(i) value of 1.54 microM (14, P1 Abu-OH). The electrophilic inhibitors exhibit increased potency as compared with the corresponding product inhibitor, and notably also the non-electrophilic P1 alkenyl alcohol 10e. This represents the first example of non-electrophilic inhibitors that are not P1 amides or product inhibitors.     

Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.     

Time-dependent inhibition of protein farnesyltransferase by a benzodiazepine peptide mimetic

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the prenylation of proteins with a carboxy-terminal tetrapeptide sequence called a CaaX box, where C refers to cysteine, "a" refers to an aliphatic residue, and X typically refers to methionine, serine, or glutamine (FTase), or to leucine (GGTase-I). Marsters and co-workers [(1994) Bioorg. Med. Chem. 2, 949--957] developed inhibitors of FTase with cysteine and methionine attached to an inner hydrophobic benzodiazepine scaffold. We found that the most potent of these compounds (BZA-2B) resulted in the time-dependent inhibition of FTase. The K(i) of BZA-2B for FTase, which is the dissociation constant of the initial complex, was 79 +/- 13 nM, and the K(i)*, which is the overall dissociation of inhibitor for all enzyme forms, was 0.91 +/- 0.12 nM. The first-order rate constant for the conversion of the initial complex to the final complex was 1.4 +/- 0.2 min(-1), and that for the reverse process was 0.016 +/- 0.002 min(-1). The latter rate constant corresponds to a half-life of the final complex of 45 min. Our experiments favor the notion that the inhibitor binds to the FTase--farnesyl diphosphate complex which then undergoes an isomerization to form a tighter FTase*--farnesyl diphosphate--BZA2-B complex. Diazepam, a compound with a benzodiazepine nucleus but lacking amino acid extensions, was a weak (K(i) = 870 microM) but not time-dependent inhibitor of FTase. Cys-Val-Phe-Met and Cys-4-aminobenzoyl-Met were instantaneous and not time-dependent inhibitors of FTase. Furthermore, BZA-4B, with a leucine specificity determinant, was a classical competitive inhibitor of GGTase-I and not a time-dependent inhibitor.     

Characterization of novel imidazole derivative, JM-8686, a potent inhibitor of allene oxide synthase

The inhibitory properties of a first synthetic jasmonic acid biosynthesis inhibitor, JM-8686, were investigated. Steady-state kinetic analysis indicates that the compound is a competitive inhibitor of allene oxide synthase (AOS) with a K(i) value of approximate 0.62+/-0.15 microM. Dialysis experiment indicates that AOS inactivation by JM-8686 is reversible. The optical difference spectroscopy analysis of JM-8686 and AOS interaction indicates that JM-8686 induced type II binding spectra with a K(d) value of approximate 1.6+/-0.2 microM, suggesting that JM-8686 binds to the prosthetic heme iron of AOS. Comparison of the inhibitory potency of the compound against HPL (CYP74B) from tomato revealed that JM-8686 was a highly selective inhibitor for AOS.     

Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method

I report herein two 2.0 ns (1.0 fs time step) MD simulations of two zinc complexes bridged by a hydroxide in phosphotriesterase (PTE) employing the nonbonded method and the cationic dummy atom method that uses virtual atoms to impose orientational requirement for zinc ligands. The cationic dummy atom method was able to simulate the four-ligand coordination of the two zinc complexes in PTE. The distance (3.39 +/- 0.07A) between two nearby zinc ions in the time-average structure of PTE derived from the MD simulation using the cationic dummy atoms matched that in the X-ray structure (3.31 +/- 0.001A). Unequivocally, the time-average structure of PTE was able to fit into the experimentally determined difference electron density map of the corresponding X-ray structure. The results demonstrate the practicality of the cationic dummy atom method for MD simulations of zinc proteins bound with multiple zinc ions. In contrast, a 2.0 ns (1.0 fs time step) MD simulation using the nonbonded method revealed a striking difference in the active site between the X-ray structure and the time-average structure that was unable to fit into the density map of PTE. The results suggest that caution should be used in the MD simulations using the nonbonded method.     

Inhibition of rat testis microsomal 3beta-hydroxysteroid dehydrogenase activity by tributyltin

In this study, we have examined the effects of a range of organotin compounds (mono-, di-, tributyltin, mono-, di-, trioctyltin) on the activities of rat testis microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxylase (17-OHase) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). 17-OHase activity was inhibited by more than 50% compared with the control rate by 59 microM tributyltin (TBT) but other organotin compounds showed no inhibition. 17beta-HSD activity was unaffected by all organotins tested. 3beta-HSD was inhibited by monooctyltin (81 microM) and by TBT at all concentrations tested in a dose-dependent manner, with almost complete loss of activity at TBT concentrations of 12 microM. The mechanism of inhibition of 3beta-HSD was investigated in kinetic analysis with 0-12 microM TBT. Three rat testis microsomal preparations were incubated with dehydroepiandrosterone as the steroid substrate ranging from 1 to 10,000 nM. Tributyltin was primarily a competitive inhibitor of 3beta-HSD activity, causing an increase in the value of the K(m(app)). However, the mechanism was not entirely competitive as while there was an increase in K(m(app)), a decrease in the V(max(app)) was also observed with increasing concentrations of TBT. Slope and intercept replots demonstrated that the K(i)((app)) from slope replots was around 2.7 microM whereas the K(i)((app)) value from intercept replots was around 30 microM. When compared with the K(m(app)) for 3beta-HSD of around 0.42 microM, TBT could be an effective inhibitor of this enzyme.     

Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase

Inhibition of lipoxygenase (LO) is currently an important goal of biomedical research due to its critical role in asthma, atherosclerosis, and cancer regulation. Steady-state kinetic data indicate that oleic acid (OA) is a simple competitive inhibitor for soybean lipoxygenase; however, kinetic isotope effect (KIE) data suggest a more complicated inhibitory mechanism. To investigate the inhibitory effects of fatty acids on lipoxygenase more thoroughly, we have synthesized a novel inhibitor to lipoxygenase, (Z)-9-octadecenyl sulfate (oleyl sulfate, OS), which imparts kinetic properties that are inconsistent with simple competitive inhibition for both SLO-1 and 15-HLO. The KIE exhibits a hyperbolic rise with addition of OS, indicating the formation of a catalytically active ternary complex with K(D) values of 0.6 +/- 0.2 and 0.4 +/- 0.05 microM for SLO-1 and 15-HLO, respectively. The steady-state kinetics show that SLO-1 proceeds through a hyperbolic mixed-type inhibition pathway, where OS binding (K(i) = 0.7 +/- 0.3 microM) causes an approximate 4-fold increase in the K(m)(app) (alpha = 4.6 +/- 0.5) and a decrease in the k(cat) by approximately 15% (beta = 0.85 +/- 0.1). 15-HLO also exhibits a hyperbolic saturation of k(cat)/K(m) consistent with the observed rise in its KIE. Taken together, these findings indicate the presence of an allosteric site in both SLO-1 and 15-HLO and suggest broad implications regarding the inhibition of LO and the treatment of LO-related diseases.     

Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) 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 lambdaPP, albeit with inhibition constants (K(i)) 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 (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP 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 Mn(2+) 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+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.