Bovine inositol monophosphatase: enzyme—metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy

Abstract

Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between inositol monophosphatase and Mg2+ ions. The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM). The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased. This is mediated almost entirely by change in the rate kdiss. A slow increase occurs in the fluorescence intensity of the pyrene-labelled enzyme after the initial, fast, increase in fluorescence caused by the binding of the Mg2+ ion. The rate of this change is independent of the concentration of the metal ion, implying that it may be a structural change in the enzyme-Mg2+ complex. Neither the fast nor the slow change in fluorescence intensity occurs when enzyme subjected to limited proteolysis by trypsin, which removes the N-terminal 36 residues, is mixed with Mg2+ ions. The data suggest that interaction with Mg2+ ions at a high-affinity site leads to a structural change in inositol monophosphatase. The data further confirm the importance of the presence of two metal ions in the structure/function of this enzyme, and show that the binding of the metal ions is not competitive with that of H+ ions and that the variation in Kd with pH is mediated almost totally by changes in kdiss.

Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

“…The effect found for pH on the affinity of Mg2 + for site 1 is in close agreement with values obtained by stopped-flow fluorescence studies [18] suggesting that the same site is examined by both techniques, although the latter studies used pyrene-labelled protein. The effect of pH on the affinity of Mg" at site 2 is very close to those found for the effect of pH on the K, for this ion [7] supporting the concept that the signal changes observed at 220 nm are due to metal ion interaction at site 2, i.e.…”

Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme

“…We analyzed the event I by performing one independent MSM analysis per subunit (see Methods), which provided remarkably consistent kinetic and affinity estimations between the monomers ( Table 2 ). In quantitative terms, the standard free energy of binding is computed to be −3.8 ± 0.1 kcal/mol, slightly higher than previous dialysis (−4.8 kcal/mol) 22 and fluorescence (−4.6 kcal/mol) 34 experiments. There are no available crystal structures containing only one Mg 2+ bound, however, in binary or ternary complexes Mg-I appears coordinated to three protein residues (Glu70, Asp90, Ile92) and three water molecules.…”

“…Only in 4 trajectories out of more than 5000, Mg-I evolved to the crystallographic position with an RMSD lower than 1 Å. Thorne et al . performed stopped-flow fluorescence spectroscopy studies to determine association and dissociation constants for Mg-I and II 34 . The study showed a slow increase in fluorescence after a rapid binding of Mg-I, suggesting that Mg-I binding is followed by a subtle structural rearrangement in the microenvironment of site I.…”

Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase

“…Numerous approaches, such as enzyme kinetics , site‐directed mutagenesis (e.g. H217Q and C218A) and spectroscopic studies , have been performed to characterize the Li + ion binding site in IMPase. It has been proposed that the Mg2 site is responsible for Li + inhibition.…”