SPECTROSCOPIC STUDIES OF MYOINOSITOL MONOPHOSPHATASE WITH A NOVEL FLUORESCENT SUBSTRATE

myo-Inositol monophosphatase catalyzes dephosphorylation of the synthe tic substrate antranyloyl-2'-AMP. Binding of this fluorescent substrat e to Tb(III)-monophosphatase was monitored by luminescence spectroscop y. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm . Assuming a mechanism of radiationless energy transfer, the actual di stance of separation between the donor anthraniloyl moiety and the acc eptor Tb(III) was calculated to be R = 10 Angstrom. The binding studie s support the earlier observation of Bone et al. (Proc. Natl. Acad. Sc i. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd (III) interact with a common binding domain of the protein. The cataly tic activity of the monophosphatase is completely dependent upon Mg(II ) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.