Chemical and kinetic mechanism of the inositol monophosphatase reaction and its inhibition by Li⁺

Abstract

Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li'], Li' inhibited the hydrolysis of racemic myo-inositol 1 -phosphate, myo-inositol4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent K, values of 1.1, 0.11 and 1.52mM, respectively. At Li' concentrations higher than 4 mM, Li' acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, K, greater than 1.5 mM.The enzyme was unable to catalyze the transesterification of ['4C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li' binds after P-0 bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of "0 from water into phosphate was shown to be completely dependent upon inositol. The K, for inositol for "0 exchange was 190 mM and in the presence of saturating phosphate, VEX was at least 60% of V,, for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li' exerts its action by binding to enzyme/product complexes. At low concentration, Li' inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol I-phosphate and 2'AMP as the substrate but not with inositol4-phosphate. The combined results indicate that Mgz' and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (K, = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li' and phosphate, increasing [Li'] caused the K, for phosphate to decrease by a factor of (1 + [Li+]/K,,). The K, for the first product off (inositol) was, however, unaltered by Li'. The results indicate that Li' can bind to the species E.Ins.P, and E.P,, but not to enzyme/substrate complexes. Further examination of the burst-phase release of [14C]inositol and its rate relative to that of the steady-state reaction under a variety of conditions revealed that Li' acts as a retarder rather than as a dead-end inhibitor and that the burst was due to hysteresis.Evidence is provided to suggest that Mg2+ is required for the catalysis only and that Li' occupies the site vacated by Mg2+ in its action as an inhibitor. The mechanisms of the reactions, the modes of inhibition...

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

“…The N-terminal signal peptide, which in vivo targets MtHPP to chloroplast, was determined by the TargetP version 1.1 server (45,46). The cleavage was predicted to occur between Arg 51 and Ala 52 , and the overexpressed construct was designed to yield the final protein with sequence starting from Met 53 . Enzymatic tests revealed that indeed, in the presence of Mg 2ϩ , MtHPP was able to hydrolyze HOLP to HOL, with the release of inorganic phosphate.…”

“…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).…”

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 and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…The phosphatase reaction of Li ϩ -sensitive phosphatases has an absolute requirement for metal ions and has been suggested to proceed through the activation of a water molecule and its subsequent nucleophilic attack on the removable phosphorus atom (57,58). It is still not clear how many metal ions are required for the activation of the nucleophilic water molecule, and two catalytic models have been proposed.…”