Mechanism of inositol monophosphatase, the putative target of lithium therapy.

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 Ihibition 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 ofMn2+, 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, stabiing 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.In the phosphatidylinositol second-messenger pathway, receptor-activated phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate, to form two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ and protein kinase C-mediated signal transduction, respectively (1-3). Inositol 1,4,5-trisphosphate is subsequently metabolized in a series of pathways, regenerating inositol for reincorporation into inositol phospholipids, thereby maintaining this signaling cycle. myo-Inositol monophosphatase (IMPase; myo-l-phosphate phosphohydrolase, EC 3.1.3.25) catalyzes the last hydrolytic step in these pathways, regenerating myo-D-inositol from myo-D-inositol 1-phosphate [Ins(l)P], Ins(3)P, or Ins(4)P (4).IMPase, a homodimer of two 30-kDa subunits, requires Li+ parameters gave contacts in agreement with those seen in the Cambridge Crystallographic Database (13), but to achieve similarly satisfactory results the van der Waals radius parameter of Gd3+ was reduced to 2.1 A. Hydrogens were added to the protein by using the Sybyl BIOPOLY ADDH command. The standard Sybyl TIP3P water model was used for crystallographically observed water molecules. Charges were taken from the standard Kollman parameter set for water molecules and protein residues. Starting coordinates for dianionic Ins(l)P were obtained from the crystal structure of the IMPase/Gd/Ins(l)P complex, supplemented with hydrogens at idealized sites on the inositol moiet...

An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings

“…We have named this unusual phenomenon ' uncompetitive activation ', in analogy to ' uncompetitive inhibition ', in which an inhibitor decreases V max and K m values in parallel. Two examples of uncompetitive inhibition are the inhibition of mammalian glucose-6-phosphate dehydrogenase by dehydroepiandrosterone [29] and the inhibition of myo-inositol monophosphatase by lithium [30]. The rarity of this type of inhibition has been ascribed to its potentially catastrophic consequences for a metabolic system [31] Most plant myrosinases are activated to some degree by ascorbate.…”

Occurrence of myo-inositol-1-phosphate phosphatase in pteridophytes: characteristics of the enzyme from the reproductive pinnules of Dryopteris filix-mas (L.) Schott

“…Chhetri et al (2005Chhetri et al ( , 2006 have already established the occurrence of the prime enzyme of myo-inositol biosynthesis, MIPS, in pteridophytes. Therefore, the presence of MIPP adds (Eisenberg Jr. 1967, Attwood et al, 1988Gee et al, 1988;Honchar et al, 1989;Leech et al, 1993;Pollack et al, 1994;Caselli et al, 1996, Fujimoto et al, 1996, Nigou and Besra, 2002Wang et al, 2006;Patra et al, 2007). However, its kinetic parameter, thermo-tolerance and calciumdependent inhibition at lower concentrations, differ substantially in pteridophytic species.…”

“…In pteridophytes, Benaroya et al (2004) and Chettri et al (2005Chettri et al ( , 2006 have recently documented the occurrence and characterization of L-myo-inositol-1-phosphate synthase. So far, work with regard to the participation of the subsequent enzyme in this metabolic sequence, myo-inositol-1-phosphate phosphatase (MIPP), has been carried out principally in animal systems (Eisenberg Jr, 1967;Attwood et al, 1988;Gee et al, 1988;Honchar et al, 1989;Leech et al, 1993;Pollack et al, 1994;Kwok and Lo, 1994;Fujimoto et al, 1996;Caselli et al, 1996), in archaea (Wang et al, 2006), in bacteria (Nigou and Besra, 2002), and in cyanobacteria (Patra et al, 2007). However, only a few reports are available of this enzyme from plant systems (Loewus and Loewus, 1983;Gumber et al, 1984).…”

Structural Studies of Medicago truncatula Histidinol Phosphate Phosphatase from Inositol Monophosphatase Superfamily Reveal Details of Penultimate Step of Histidine Biosynthesis in Plants

“…It is therefore very likely that only Mg 2ϩ binding site 1 with K d Ϸ 300 M (68) and site 2 with K d Ϸ 3 mM (69) are occupied in vivo. More importantly, Mg 2ϩ bound at site 1 was reported to remain tethered throughout the entire catalytic cycle, unlike Mg 2ϩ at site 2 that binds and dissociates over the course of the reaction (53,61,62).…”