Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

Abstract

Mg" 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 ( K J around 275 pM, 292 pM and 302 pM 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 Mgz+ 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 Mg" 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 P,, there is a fourfold increase in the affinity of wild-type enzyme for Mg".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 3 ) . 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 K,, value for this metal ion.Other metal ions such as Ca2+ and Tb" (but not Mn'+ or Znz+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg" or Tb" (25%) ions than for Ca2+ (8.5%), although two Caz+-binding sites with Kd values of 20 pM and 100 pM have been identified.The uncompetitive inhibitor Li' causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or P,. 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.Keywords: inositol monophosphatase ; circular dichroism; lithium ; magnesium.Inositol monophosphatase is a homodimeric enzyme of subunit molecular mass 30 kDa that catalyses the dephosphorylation of Ins( 1)P, Ins(3)P and Ins(4)P (inositol monophosphates) to liberate inositol and P, (inorganic phosphate), the former of which may be used to replenish stores of membrane-bound phosphatidylinositol 4,5-bisphosphate [l]. The pivotal role in the phosphoinositol lipid signalling pathway played by this enzyme is of greater significance in the brain due to the inability of dietary inositol to cross the blood-brain barrier effectively 121, although inositol may be synthesised de novo from glucose 6-phosphate via the action of inositol 1-synthase [3]. A high level of sequence similarity between the human, rat and bovine forms of the enzyme has been demonstrated using cDNA cloning and sequencing techniques [4, 51. Mg2' ions are absolutely required for activity [6], although concentratio...

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

“…These results are in contrast to mammalian IMPase where the far-UV CD spectrum was sensitive to mM Mg 2þ and showed a significant loss of secondary structure with a 'K D ' of 4 mM. 26 The latter could reflect structural changes related to the kinetic inhibition at higher Mg 2þ that is seen with the mammalian IMPase but not with archaeal IMPase enzymes. Fig.…”

X-ray structure of yeast hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity

“…By analogy to the situation in Hal2p, the effect of the I68A mutation must be mediated by the altered hydrophobic interaction with Trp219 affecting the position of active site Asp220. It is plausible that the three previously described mutations of HAPase affecting cation sensitivity (H217Q, C218A and W219F) (Gore et al, 1993;Rees-Milton et al, 1997) also act by displacing Asp220.…”

myo-Inositol Monophosphatase: A Challenging Target for Mood Stabilising Drugs

“…It was concluded that binding of Mg 2+ at the high affinity site (site 1) is accompanied by a pronounced conformational change in the enzyme [49]. The latter binding constant was strengthened four fold by the presence of phosphate [51]. The results again gave evidence for structural fluctuation of IMPase upon metal ion binding and also showed that more than one metal ion is required for catalysis [50].…”

“…Furthermore, a slow change in fluorescence was observed, that occurred after the fast initial mixing and was independent of metal binding; when the first 36 N-terminal residues were removed by limited proteolysis, neither the fast nor the slow fluorescence change occurred. The effects of metal binding upon the CD spectrum of IMPase have also been studied [51]. Kwon and Churchich used a pyrene-IMPase derivative and an eosin-IMPase derivative to study Mg 2+ , Co 2+ and Tb 3+ binding to IMPase through observation of changes in the fluorescent and phosphorescent spectra upon metal ion binding respectively [50].…”

“…Kwon and Churchich used a pyrene-IMPase derivative and an eosin-IMPase derivative to study Mg 2+ , Co 2+ and Tb 3+ binding to IMPase through observation of changes in the fluorescent and phosphorescent spectra upon metal ion binding respectively [50]. Li + only perturbed the CD spectrum at 220 nm, implying once more that it inhibits the enzyme by binding at metal site 2 [51]. The effects of metal binding upon the CD spectrum of IMPase have also been studied [51].…”