Myo-Inositol Monophosphatase: Binding of Terbium and a Cross-Linking Reagent to the Catalytic Site Cavity

In the presence of myo-inositol monophosphatase, terbium ions can be excited by energy transfer from the aromatic side chains of the protein. This enhancement of Tb3+ luminescence due to its binding to the enzyme at pH 6.5 was used to determine the dissociation constant (56 μM) for the monophosphatase-Tb3+ complex. Competition luminescence studies also indicated that calcium ions could compete with terbium ions for binding to the enzyme with a dissociation constant of 200 μM. Conditions were determined in which approximately 1 mol of o-phthaldehyde reacts with one subunit of the dimeric protein, yielding a stable fluorescent isoindole derivative. The kinetics of the reaction, monitored by fluorescence spectroscopy, yields a second-order rate constant (K2 = 110 M−1 s−1). The inactivation of the monophosphatase by o-phthaldehyde has been shown to be protected by the substrate β-glycerolphosphate. Essentially, no formation of any isoindole derivative is detected after one sulfhydryl residue of the enzyme is reacted with N-ethylmaleimide. It is suggested that the site of the isoindole chromophore formed upon reaction of the enzyme with o-phthaldehyde includes one lysyl and one cysteinyl residue located in the cavity of the catalytic site.