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 region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA 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 (Kd) around 275 microM, 292 microM and 302 microM 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 decrease in ellipticity over most of the far-ultraviolet region of the spectrum (between 205-240 nm). The Kd values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12% change in ellipticity. In the additional presence of 10 mM Pi, there is a fourfold increase in the affinity of wild-type enzyme for Mg2+. It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 1). The spectral changes around 225 nm are associated with interactions at a lower-affinity site normally occupied by the Mg2+ ion which is reflected by the Km 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 changes are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), although two Ca2+-binding sites with Kd values of 20 microM and 100 microM have been identified. The uncompetitive inhibitor Li+ causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or Pi. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.