The binding of substrates and inhibitors to the metal center of myoinositol monophosphatase.
The synthetic substrate anthraniloyl-beta-glycerol-P binds to myoinositol monophosphatase with a Kd = 5 microM at pH 7.5. The anthraniloyl chromophore, excited at 330 nm, sensitizes the long lived luminescence of bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor-acceptor pair was calculated to be R = 10 A. Tb(III) binds to the monophosphatase with a Kd = 2 microM, whereas Ca-(II) displaces the lanthanide at concentrations above 0.1 mM. The binding studies support the notion that Tb(III), Ca(II) and Mg(II) interact with a common binding site on the protein. Phosphate ion, a strong competitive inhibitor, perturbs the luminescence of bound Tb(III), whereas the substrate beta-glycero-P has no effect on the luminescence yield and long-lived emission of bound Tb(III). It is suggested that the phosphate group of the substrate is not in direct contact with the metal ion coordinated to several amino acid residues of the enzyme.
Kwok
F
F
Department of Applied Biology and Chemistry Technology, Hong Kong Polytechnic.
Churchich
J E
JE
D062365:Aminobenzoates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D005994:Glycerophosphates / Q000378:metabolism* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000494:pharmacology; D049449:Luminescence; D010710:Phosphates / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013725:Terbium / Q000378:metabolism*
D062365:Aminobenzoates; D005994:Glycerophosphates; D007295:Inositol Phosphates; D010710:Phosphates; D013725:Terbium; C073394:2-aminobenzoyl-glycerophosphate; C002647:inositol 1-phosphate; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D002118:Calcium; C031463:beta-glycerophosphoric acid