High affinity association of myo-inositol trisphosphates with phytase and its effect upon the catalytic potential of the enzyme.
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 ( approximately 66 kDa) contains the catalytic site, and the smaller peptide ( approximately 5 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.
Padmanabhan
U
U
Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Kolkata 700 037, India.
Dasgupta
S
S
Biswas
B B
BB
Dasgupta
D
D
D010832:6-Phytase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D007295:Inositol Phosphates; D010832:6-Phytase; D014357:Trypsin; D002118:Calcium