DETECTION OF METAL-BINDING TO BOVINE INOSITOL MONOPHOSPHATASE BY CHANGES IN THE NEAR AND FAR-ULTRAVIOLET REGIONS OF THE CD SPECTRUM

Mg2+ ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near-ultraviolet regi on of the CD spectrum of the enzyme. These spectral changes are not af fected by the additional presence of substrate and are reversed if EDT A is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant (K-d) around 275 mu M, 292 mu M and 302 mu M for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219. The addition of Mg2+ also causes a dec rease in ellipticity over most of the far-ultraviolet region of the sp ectrum (between 205-240 nm). The K-d values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]in ositol monophosphatase and [Phe219]inositol monophosphatase enzymes, r espectively, each showing an approximate 12% change in ellipticity. In the addition presence of 10 mM P-i, there is a fourfold increase in t he affinity of wild-type enzyme for Mg2+. It is concluded that CD spec tral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 1). The spe ctral changes around 225 nm are associated with interactions at a lowe r-affinity site normally occupied by the Mg2+ ion which is reflected b y the K-m value for this metal ion. Other metal ions such as Ca2+ and Tb3+ (but not Mn2+ or Zn2+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal chang es are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), altho ugh two Ca2+-binding sites with K-d values of 20 mu M and 100 mu M hav e been identified. The uncompetitive inhibitor Li+ causes little chang e in the near-ultraviolet spectrum in the absence or presence of eithe r substrate or P-i. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a K-d of 0.8 nM.