The regulation of myo-inositol-1-phosphate-synthase activity from Neurospora crassa by pyrophosphate and some cations

Abstract

The effect of several cations on the inhibition of PP i of the enzyme myo-inositol-1-phosphate synthase ( 1 l -myo- inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4) from Neurospora crassa was studied. The study was undertaken in an attempt to explain how myo-inositol biosynthesis can occur in the presence of an intracellular PP i concentration which exceeds the K i for the enzyme by 350-fold. The inhibition of enzyme activity by PP i, at pH 7.7, was reversed, in decreasing order of effectiveness, by Mn 2+, Fe 3+, Fe 2+, Mg 2+, Co 2+, Cu 2+, Ca 2+, Ba 2+ and Zn 2+. The concentration of Mg 2+ shown to be effective in reversing inhibition is well within the range demonstrable in N. crassa mycelia. It was shown that inhibition of the enzyme by PP i occurs only in the presence of NH + 4, an activator of the enzyme, and that inhibition is pH dependent. The data presented suggest that the in vivo regulation of myo-inositol biosynthesis occurs as a consequence of the modulation of myo-inositol-1-phosphate synthase activity by PP 1, pH, and several cations. myo-Inositol-1-phosphate synthase ( 1 l -myo- inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4), catalyzes the synthesis of l -myo- inositol-1-P from d - glucose-6-P with an absolute requirement for NAD +. Previously it was reported that with a partially-purified enzyme preparation from N. crassa, PP i acts as a competitive inhibitor and that the enzyme has an apparent K m of 1.9 mM and a K i of 4.6 μM [1]. In addition, the suggestion was made that PP i might contribute to the regulation of myo-inositol (inositol) biosynthesis in N. crassa. We subsequently presented experimental evidence in favor of a regulatory role for PP i in the in vivo biosynthesis of inositol by showing that a four-fold decrease in the PP i pool is accompanied by a four to five-fold increase in the rate of conversion of [U- 14C] glucose to [ 14C] inositol [2]. Determination of the PP i pool in wild-type myclia, gave a value of 6.29 μmol/g dry weight or approximately 1.7 mM, assuming 4 ml of intracellular water per g dry weight of cells [2]. However, since the intracellular PP i concentration is 350-fold greater than the K i value for the enzyme, it is difficult to understand how inositol can readily be synthesized in vivo. This report deals with a more detailed study of the factors which influence the inhibition of myo-inositol-1-phosphate synthase by PP i, in order to better understand the regulation of inositol biosynthesis in N. crassa.