MYOINOSITOL 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 e xcited by energy transfer from the aromatic side chains of the protein . This enhancement of Tb3+ luminescence due to its binding to the enzy me at pH 6.5 was used to determine the dissociation constant (56 mu M) for the monophosphatase-Tb3+ complex. Competition luminescence studie s also indicated that calcium ions could compete with terbium ions for binding to the enzyme with a dissociation constant of 200 mu M. condi tions were determined in which approximately 1 mol of o-phthaldehyde r eacts with one subunit of the dimeric protein, yielding a stable fluor escent isoindole derivative. The kinetics of the reaction, monitored b y fluorescence spectroscopy, yields a second-order rate constant (K-2 = 110 M(-1) s(-1)). The inactivation of the monophosphatase by o-phtha ldehyde has been shown to be protected by the substrate beta-glycerolp hosphate. Essentially, no formation of any isoindole derivative is det ected after one sulfhydryl residue of the enzyme is reacted with N-eth ylmaleimide. It is suggested that the site of the isoindole chromophor e formed upon reaction of the enzyme with o-phthaldehyde includes one lysyl and one cysteinyl residue located in the cavity of the catalytic site. (C) 1994 Academic Press,Inc.