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John R. Atack - One of the best experts on this subject based on the ideXlab platform.

  • crystallization and structure of ebselen bound to cys141 of human Inositol Monophosphatase
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2020
    Co-Authors: Gareth D Fenn, John R. Atack, Helen Wallerevans, Benjamin Bax
    Abstract:

    Inositol Monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47 A resolution crystal structure (PDB entry 6zk0), are presented. In the complex with human IMPase, ebselen in a ring-opened conformation is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and in the crystal structure two adjacent dimers share four ebselen molecules, creating a tetramer with approximate 222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor or may block the binding of partner proteins.

  • Co-crystallization of human Inositol Monophosphatase with the lithium mimetic L-690,330
    Acta Crystallographica Section D Structural Biology, 2018
    Co-Authors: Lucas Kraft, S. Mark Roe, R. Gill, John R. Atack
    Abstract:

    Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit Inositol Monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by attenuating phosphatidylInositol (PI) cell signalling. Drug-discovery efforts have focused on small-molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L-690,330 is a potent bisphosphonate substrate-based inhibitor developed by Merck Sharp & Dohme. To aid future structure-based inhibitor design, determination of the exact binding mechanism of L-690,330 to IMPase was of interest. Here, the high-resolution X-ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 A resolution is reported.

  • Inositol Monophosphatase inhibitors--lithium mimetics?
    Medicinal research reviews, 1997
    Co-Authors: John R. Atack
    Abstract:

    Given the putative role of Inositol Monophosphatase (IMPase) as the molecular target for the therapeutic effects of lithium, inhibitors of this enzyme have been proposed to be lithium-mimetics. Although cation and natural product inhibitors of IMPase have been described, these have not proved suitable for cell culture studies due to a lack of specificity. On the other hand, substrate (Inositol 1-phosphate)-based inhibitors have proved useful for showing that this class of compounds mimic lithium with respect to effects on the phosphatidylInositol (PI) cell-signaling pathway. However, since these compounds are highly charged, their polarity means they are not suitable for in vivo use. Finally, the recent elucidation of the structure and mechanism of IMPase may provide the opportunity to develop compounds which specifically interact with key structural and mechanistic features of the enzyme and would represent novel structure- or mechanism-based inhibitors.

  • Inositol Monophosphatase, the putative therapeutic target for lithium
    Brain research. Brain research reviews, 1996
    Co-Authors: John R. Atack
    Abstract:

    Lithium has been hypothesised to exert its therapeutic effects in the treatment of bipolar disorder by attenuating phosphatidylInositol (PI) cell signalling pathways that are presumably hyperactive in this disorder. More specifically, lithium has been proposed to inhibit Inositol Monophosphatase (IMPase) thereby causing a depletion of intracellular Inositol which results in a reduction in the synthesis of the PI required to sustain this signalling pathway. In the present article this 'Inositol depletion' hypothesis will be reviewed and pathological, pharmacodynamic, developmental and anatomical aspects of IMPase as well as inhibitors of this enzyme will be described.

  • Inositol Monophosphatase a putative target for li in the treatment of bipolar disorder
    Trends in Neurosciences, 1995
    Co-Authors: John R. Atack, Howard B. Broughton, Scott J. Pollack
    Abstract:

    Abstract Attenuation of the phosphatidylInositol (PI) signal transduction pathway as a consequence of inhibition of Inositol Monophosphatase (IMPase) has been proposed as the mechanism for the efficacy of Li + in the treatment of bipolar disorder. Nevertheless, Li + also affects other aspects of PI signal transduction, and it is therefore not clear whether modulation of PI responses by Li + can be attributed solely to inhibition of IMPase. However, inhibitors of IMPase as target of Li + on some aspects of PI cell signalling, thus highlighting the potential of IMPase as a target for the treatment of bipolar disorder. The recent description of the three-dimensional structure of IMPase in conjunction with site-directed mutagenesis and kinetic studies has led to the elucidation of the enzyme mechanism. These structural and mechanistic data should prove useful in the development of novel inhibitors of IMPase that might ultimately prove useful clinically.

C I Ragan - One of the best experts on this subject based on the ideXlab platform.

  • structural analysis of Inositol Monophosphatase complexes with substrates
    Biochemistry, 1995
    Co-Authors: Roger Bone, C I Ragan, James P. Springer, John R. Atack, Scott J. Pollack, Michael R. Knowles, George Mcallister, L Frank, S A Osborne, H.b. Broughton
    Abstract:

    The structures of ternary complexes of human Inositol Monophosphatase with inhibitory Gd3+ and either D- or L-myo-Inositol 1-phosphate have been determined to 2.2-2.3 A resolution using X-ray crystallography. Substrate and metal are bound identically in each active site of the phosphatase dimer. The substrate is present at full occupancy, while the metal is present at only 35% occupancy, suggesting that Li+ from the crystallization solvent partially replaces Gd3+ upon substrate binding. The phosphate groups of both substrates interact with the phosphatase in the same manner with one phosphate oxygen bound to the octahedrally coordinated active site metal and another oxygen forming hydrogen bonds with the amide groups of residues 94 and 95. The active site orientations of the Inositol rings of D- and L-myo-Inositol 1-phosphate differ by rotation of nearly 60 degrees about the phosphate ester bond. Each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. Mutagenesis experiments confirm the interaction of Glu 213 with the Inositol ring and suggest that interactions with Ser 165 may develop during the transition state. The structural data suggest that the active site nucleophile is a metal-bound water that is activated by interaction with Glu 70 and Thr 95. Expulsion of the ester oxygen appears to be promoted by three aspartate residues acting together (90, 93, and 220), either to donate a proton to the leaving group or to form another metal binding site from which a second Mg2+ coordinates the leaving group during the transition state.

  • Mechanism of Inositol Monophosphatase, the putative target of lithium therapy
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Scott J. Pollack, C I Ragan, John R. Atack, Michael R. Knowles, George Mcallister, R. Baker, S.r. Fletcher, Leslie L. Iversen, H.b. Broughton
    Abstract:

    Abstract myo-Inositol Monophosphatase (myo-Inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylInositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human Inositol Monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabilizing the leaving Inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.

  • Bovine Inositol Monophosphatase: The identification of a histidine residue reactive to diethylpyrocarbonate
    FEBS letters, 1993
    Co-Authors: Karen Rees-milton, C I Ragan, Peter J. Greasley, Michael G. Gore
    Abstract:

    Abstract The Inositol Monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217→Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.

  • Bovine Inositol Monophosphatase: proteolysis and structural studies
    FEBS Letters, 1993
    Co-Authors: Peter J. Greasley, Michael G. Gore, Karen Rees-milton, C I Ragan
    Abstract:

    Bovine brain Inositol Monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of α helices. The most susceptible bond (Lys-36-Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97-120].

  • cDNA cloning of human and rat brain myo-Inositol Monophosphatase. Expression and characterization of the human recombinant enzyme.
    The Biochemical journal, 1992
    Co-Authors: G Mcallister, P Whiting, E A Hammond, M R Knowles, J R Atack, F J Bailey, R Maigetter, C I Ragan
    Abstract:

    Inositol Monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide Inositol required for the resynthesis of phosphatidylInositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine Inositol Monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.

Antonella Riccio - One of the best experts on this subject based on the ideXlab platform.

  • an ngf responsive element targets myo Inositol Monophosphatase 1 mrna to sympathetic neuron axons
    Nature Neuroscience, 2010
    Co-Authors: Catia Andreassi, Carola Zimmermann, Richard Mitter, Salvatore Fusco, Adolfo Saiardi, Serena De Vita, Antonella Riccio
    Abstract:

    mRNA localization is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. To identify mRNAs localized in subcellular compartments of developing neurons, we took an original approach that combines compartmentalized cultures of rat sympathetic neurons and sequential analysis of gene expression (SAGE). Unexpectedly, the most abundant transcript in axons was mRNA for myo-Inositol Monophosphatase-1 (Impa1), a key enzyme that regulates the Inositol cycle and the main target of lithium in neurons. A novel localization element within the 3' untranslated region of Impa1 mRNA specifically targeted Impa1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to nerve growth factor (NGF). Selective silencing of IMPA1 synthesis in axons decreased nuclear CREB activation and induced axonal degeneration. These results provide insights into mRNA transport in axons and reveal a new NGF-responsive localization element that directs the targeting and local translation of an axonal transcript.

  • Corrigendum: An NGF-responsive element targets myo-Inositol Monophosphatase-1 mRNA to sympathetic neuron axons
    Nature Neuroscience, 2010
    Co-Authors: Catia Andreassi, Carola Zimmermann, Richard Mitter, Salvatore Fusco, Serena Devita, Adolfo Saiardi, Antonella Riccio
    Abstract:

    Corrigendum: An NGF-responsive element targets myo-Inositol Monophosphatase-1 mRNA to sympathetic neuron axons

Kousaku Murata - One of the best experts on this subject based on the ideXlab platform.

  • nadp h phosphatase activities of archaeal Inositol Monophosphatase and eubacterial 3 phosphoadenosine 5 phosphate phosphatase
    Applied and Environmental Microbiology, 2007
    Co-Authors: Chikako Fukuda, Shigeyuki Kawai, Kousaku Murata
    Abstract:

    NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an Inositol Monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal Inositol Monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known Inositol Monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for Inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed Inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The Inositol polyphosphate 1-phosphatase activity of archaeal Inositol Monophosphatase was considered to be compatible with the similar tertiary structures of Inositol Monophosphatase, fructose-1,6-bisphosphatase, Inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although Inositol Monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.

  • NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase
    Applied and environmental microbiology, 2007
    Co-Authors: Chikako Fukuda, Shigeyuki Kawai, Kousaku Murata
    Abstract:

    NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an Inositol Monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal Inositol Monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known Inositol Monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for Inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed Inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The Inositol polyphosphate 1-phosphatase activity of archaeal Inositol Monophosphatase was considered to be compatible with the similar tertiary structures of Inositol Monophosphatase, fructose-1,6-bisphosphatase, Inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although Inositol Monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.

Michael G. Gore - One of the best experts on this subject based on the ideXlab platform.

  • Stereochemistry at Phosphorus of the Reaction Catalyzed by myo-Inositol Monophosphatase
    Journal of medicinal chemistry, 2002
    Co-Authors: Christine M. J. Fauroux, Michael G. Gore, Michael Lee, Paul M. Cullis, Kenneth T. Douglas, Sally Freeman
    Abstract:

    myo-Inositol Monophosphatase (IMPase), the proposed target for lithium therapy for manic depression, is an important enzyme in the biosynthesis of second messengers. Earlier studies have shown that the IMPase-catalyzed hydrolysis of myo-Inositol monophosphates to inorganic phosphate and myo-Inositol proceeds by direct attack of water at phosphorus. However, research groups have independently proposed either an in-line displacement (with inversion of stereochemistry at phosphorus) or an adjacent attack with a pseudorotation (with retention of stereochemistry at phosphorus). Here, the elucidation of the stereochemical pathway is presented. The IMPase-catalyzed hydrolysis of D-1-Sp-myo-Inositol [17O]-thiophosphate in the presence of H218O gave inorganic Rp-[16O,17O,18O]-thiophosphate, with inversion of configuration at phosphorus. This is only consistent with an in-line displacement, and it rules out the controversial adjacent/pseudorotation mechanism. This result will assist in the design of alternative inhibitors of IMPase.

  • Detection of metal binding to bovine Inositol Monophosphatase by changes in the near and far ultraviolet regions of the CD spectrum.
    European journal of biochemistry, 1997
    Co-Authors: Karen Rees-milton, Jorge E. Churchich, Peter J. Greasley, Mark R. Thorne, Michael G. Gore
    Abstract:

    Mg2+ ions, essential for the catalytic activity of mammalian Inositol Monophosphatase, increase the ellipticity in the near-ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant (Kd) around 275 microM, 292 microM and 302 microM for the wild-type, [Gln217]Inositol Monophosphatase and [Phe219]Inositol Monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219. The addition of Mg2+ also causes a decrease in ellipticity over most of the far-ultraviolet region of the spectrum (between 205-240 nm). The Kd values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]Inositol Monophosphatase and [Phe219]Inositol Monophosphatase enzymes, respectively, each showing an approximate 12% change in ellipticity. In the additional presence of 10 mM Pi, there is a fourfold increase in the affinity of wild-type enzyme for Mg2+. It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 1). The spectral changes around 225 nm are associated with interactions at a lower-affinity site normally occupied by the Mg2+ ion which is reflected by the Km value for this metal ion. Other metal ions such as Ca2+ and Tb3+ (but not Mn2+ or Zn2+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), although two Ca2+-binding sites with Kd values of 20 microM and 100 microM have been identified. The uncompetitive inhibitor Li+ causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or Pi. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.

  • Reversible denaturation of myo-Inositol Monophosphatase. The stability of the metal-binding loop
    European journal of biochemistry, 1996
    Co-Authors: Fermin Moreno, Michael G. Gore, Karen Rees-milton, Susana Corrales, Francisco García Blanco, Jorge E. Churchich
    Abstract:

    The unfolding of bovine brain myo-Inositol Monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-Inositol Monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The Monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.

  • Bovine Inositol Monophosphatase: enzyme-metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy.
    Biochemical Journal, 1996
    Co-Authors: Mark R. Thorne, Peter J. Greasley, Michael G. Gore
    Abstract:

    Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between Inositol Monophosphatase and Mg2+ ions. The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM). The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased. This is mediated almost entirely by change in the rate kdiss. A slow increase occurs in the fluorescence intensity of the pyrene-labelled enzyme after the initial, fast, increase in fluorescence caused by the binding of the Mg2+ ion. The rate of this change is independent of the concentration of the metal ion, implying that it may be a structural change in the enzyme-Mg2+ complex. Neither the fast nor the slow change in fluorescence intensity occurs when enzyme subjected to limited proteolysis by trypsin, which removes the N-terminal 36 residues, is mixed with Mg2+ ions. The data suggest that interaction with Mg2+ ions at a high-affinity site leads to a structural change in Inositol Monophosphatase. The data further confirm the importance of the presence of two metal ions in the structure/function of this enzyme, and show that the binding of the metal ions is not competitive with that of H+ ions and that the variation in Kd with pH is mediated almost totally by changes in kdiss.

  • Spectroscopic studies of myo-Inositol Monophosphatase with a novel fluorescent substrate.
    Biochimica et biophysica acta, 1996
    Co-Authors: Teresa Pineda, Michael G. Gore, M.j. Thorne, J. E. Churchich
    Abstract:

    myo-Inositol Monophosphatase catalyzes dephosphorylation of the synthetic substrate antranyloyl-2′-AMP. Binding of this fluorescent substrate to Tb(III)-Monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10A. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031–10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the Monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.