BOVINE INOSITOL MONOPHOSPHATASE - ENZYME-METAL-ION INTERACTIONS STUDIED BY PRE-EQUILIBRIUM FLUORESCENCE SPECTROSCOPY

摘要

Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (k(ass)) and the off-rate (k(diss)) for the equilibrium between inositol monophosphatase and Mg2+ ions. The dissociation constant (K-d) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a K-d typically around 450 mu M, close to values determined by equilibrium studies (270-300 mu M). 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 a change in the rate k(diss). 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 K-d with pH is mediated almost totally by changes in k(diss).