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Modification of myo-inositol monophosphatase by the arginine-specific reagent phenylglyoxal.

ABSTRACT: myo-Inositol monophosphatase is inhibited by the arginine-specific reagent phenylglyoxal. The rate of inactivation is decreased in the presence of Pi, a competitive inhibitor of the enzyme. The effect of Pi is dependent on the presence of Mg2+, but is unaffected by Li+, an uncompetitive inhibitor. In the absence of Mg2+, the substrate, Ins(1)P, binds to the enzyme but is not converted into products, and affords only a small degree of protection against inactivation by phenylglyoxal. Li+ had no further effect under these conditions, but in the presence of Mg2+ caused a marked potentiation of the protective effect of substrate alone. In the absence of substrate, Li+ had no effect on activation by phenylglyoxal. Incorporation of 14C-labelled phenylglyoxal showed that inactivation was associated with modification of a single arginine residue per monomer in the dimeric enzyme. These findings support a mechanism in which Li+ inhibits monophosphatase by trapping a phosphorylated enzyme intermediate and preventing its hydrolysis.

SUBMITTER: Jackson RG

PROVIDER: S-EPMC1133597 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Project description:Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

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Modification of myo-inositol monophosphatase by the arginine-specific reagent phenylglyoxal.

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