A neutral phytase from germinating mung bean (Vigna radiata) seeds dephosphorylates myo-inositol hexakisphosphate sequentially to myo-inositol. The enzyme also binds with higher affinity to myo-inositol trisphosphates (1,4,5), (2,4,5), and (1,3,4) isomers without catalysis. The high affinity complex elicits Ca 2؉ mobilization in vitro from microsomes/vacuoles via the formation of a ternary complex with the receptor for Ins(1,4,5)P 3 . As a sequel to our previous report, we have carried out a detailed characterization of the two sites and examined the mutual interactions between them. Presaturation of the high affinity site leads to an increase in the affinity of the enzyme for phytic acid and its rate of dephosphorylation as well. From the products of limited tryptic cleavage of phytase, two peptides, each with one activity, have been isolated. The larger peptide (ϳ66 kDa) contains the catalytic site, and the smaller peptide (ϳ5 kDa) has the high affinity myo-inositol trisphosphate-binding site. The interaction between the dual activities of phytase has been observed also at the level of the two peptides. A sequence homology search using N-terminal 12 amino acid residues of the 5-kDa fragment has revealed significant homology with the Homer class of proteins implicated in signaling pathways involving metabotropic glutamate receptor and myo-inositol 1,4,5-trisphosphate receptor. These results indicate a second role of phytase in Ca 2؉ mobilization during germination of mung been seed via a salvage pathway that involves allosteric activation by myo-inositol trisphosphate.Phytase (D-myo-inositol hexakisphosphate hydrolase) from mung bean seed, Vigna radiata, is a neutral phosphatase that dephosphorylates myo-inositol hexakisphosphate, InsP 6 , 1 in the following sequential way: InsP 6 3 InsP 5 3 InsP 4 3 InsP 3 3 InsP 2 3 InsP 3 Ins. It is ubiquitous in its occurrence. Its amount increases during the germination of seed (1). Phytase isolated from 72-h germinating mung bean seeds is a monomeric enzyme of molecular mass of 160 kDa with a pH optimum of 7.5 (2). A previous study (3) has demonstrated that in the sequential dephosphorylation of InsP 6 , the enzyme displays increasing affinity for the substrate, InsP n (n ϭ 1-6), with a decrease in the number of phosphate groups. Binding of the substrates at the catalytic site in phytase is characterized by dissociation constants in the micromolar range (3). This report (3) showed the presence of a high affinity (dissociation constant in the nanomolar order) non-catalytic site, specific for Ins(1,4,5)/ (2,4,5)/(1,3,4)P 3 , in phytase. Phytase-myo-inositol trisphosphate complex formed at the high affinity site forms a ternary complex with the putative receptor for Ins(1,4,5)P 3 and enhances the level of calcium release from microsomal/vacuolar suspension in vitro. The amount of calcium mobilized by the high affinity complex was about three times greater than that released by myo-inositol trisphosphate alone. Another interesting observation was that the complex formation at...