Dynamics of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor.

A significant determinant for the broad substrate specificity of the metallo-beta-lactamases from Bacteroides fragilis and other similar organisms is the presence of a plastic substrate binding site that is nevertheless capable of tight substrate binding in the Michaelis complex. To achieve these two competing ends, the molecule apparently employs a flexible flap that closes over the active site in the presence of substrate. These characteristics imply that dynamic changes are an important component of the mechanism of action of these enzymes. The backbone and tryptophan side chain dynamics of the metallo-beta-lactamase from B. fragilis have been examined using (15)N NMR relaxation measurements. Two states of the protein were examined, in the presence and absence of a tight-binding inhibitor. Relaxation measurements were analyzed by the model-free method. Overall, the metallo-beta-lactamase molecule is rigid and shows little flexibility except in loops. The flexibility of the loop that covers the active site is not unusually great as compared to the other loops of the protein. Local motion on a picosecond time scale was found to be very similar throughout the protein in the presence and absence of the inhibitor, but a significant difference was observed in the motions on a nanosecond time scale (tau(e)). Large-amplitude motions with a time constant of about 1.3 ns were observed for the flexible flap region (residues 45-55) in the absence of the inhibitor. These motions were completely damped out in the presence of the inhibitor. In addition, the motion of a tryptophan side chain at the tip of the beta-hairpin of the flap shows a very significant difference in motion on the ps time scale. These results indicate that the motions of the polypeptide chain in the flap region can be invoked to explain both the wide substrate specificity (the free form has considerable amplitude of motion in this region) and the catalytic efficiency of the metallo-beta-lactamase (the motions are damped out when the inhibitor and by implication a substrate binds in the active site).