Fluorometric Studies on the Role of Calcium in Substrate Binding to 3-Ketovalidoxylamine A C-N Lyase

In the present work, we studied the role of Ca2+ in the interaction between 3-ketovalidoxylamine A C-N lyase and its substrate in terms of the intrinsic tryptophan fluorescence of this enzyme. Intrinsic tryptophan fluorescence of the C-N lyase (1 μM), in the presence of endogenous Ca2+ (about 10 μM), was quenched by the addition of a substrate of the lyase, such as methyl-α-D-3-ketoglucoside or 3-ketotrehalose. The intensity of fluorescence returned to its original level in the absence of substrate when 0.1 mM ethylene glycol bis(2-aminoethylether)tetraacetic acid (EGTA) was added. In the presence of excess EGTA, however, the intrinsic fluorescence of the enzyme was unchanged by the addition of the substrates. The intensity of the fluorescence was also not changed by the addition of 0.1 mM CaCl2 or EGTA alone. The substrate-dependent changes in the intrinsic fluorescence completely, disappeared, showing a red shift of the emission maximum, by about 12 nm, at 0.4 M or more of guanidine HCl. These experimental results suggest that Ca2+ has an important role in the binding of the substrate to the C-N lyase by maintaining the correct microenvironment and/or conformation around the tryptophan residue(s) of the enzyme molecule. The pH-dependent profiles of the Kd for the substrate binding to the lyase and C-O lyase activity indicate that the catalytic activity is not always correlated to the ability of the substrate to bind to the enzyme.