Stereospecific recognition of inositol 1,4,5-trisphosphate analogs by the phosphatase, kinase, and binding proteins

A series of DL-inositol 1,4,5-trisphosphate (IP₃) analogs, with a bulky substituent on the 2nd carbon of the inositol ring, has been synthesized. These compounds exert biological activities with only minor reduction in potency, in several assay systems (Hirata, M., Watanabe, Y., Ishimatsu, T., Ikebe, T., Kimura, Y., Yamaguchi, K., Ozaki, S., and Koga, T. (1989) J. Biol. Chem. 264, 20303-20308). Two analogs with aminocyclohexanecarbonyl (designated as analog 206) or aminobenzoyl group (analog 209) were separated into individual optical isomers and examined for stereospecificity in recognition by IP₃-5-phosphatase, IP₃-3-kinase and IP₃ binding activity. IP₃-5-phosphatase activity of erythrocyte ghosts was competitively inhibited by L-209 with a lower K(i) value than D-IP₃, but with a higher K(i) value by L-206. D-Isomers of both analogs at 100 μM failed to inhibit the hydrolysis of D-[³H]IP₃. On the other hand, D-isomers but not L-isomers of both analogs were as potent as D-IP₃ in the recognition by IP₃-3-kinase of rat brain cytosol and only the D-isomer of analog 206 could serve as substrate for the kinase. Also D-isomers of both analogs were equipotent to D-IP₃ in displacing [³H]IP₃ binding to rat cerebellum microsomes. These observations suggest that the IP₃ analogs we synthesized are stereospecifically recognized by three IP₃-recognizable proteins, but the phosphatase recognizes opposite isomers. Such being the case, the second hydroxyl group of D-IP₃ may be involved in the recognition by IP₃-5-phosphatase, but not by IP₃-3-kinase and binding sites.