earticle

영어

Once upon binding of substrates, enzymes suffer from structural changes, i.e., from open to closed conformations. Flexible residues are closely linked to protein stability and often have an important role in conformational changes of enzymes for substrate binding. Role of flexible residues in enzyme activity could be investigated by analyzing structural differences between open and closed states. Prediction of flexible residues involved in catalytic motions is thus important not only to understand enzyme catalytic mechanism and but also to modulate activity and stability of enzymes. However, some enzymes such as xylanases do not show a distinct difference between apo and holo states. In the case of these enzymes, it is difficult to find out important flexible residues involved in structural motions with simple comparisons between two static states. In this study, we analyzed the flexibility of Bacillus circulans xylanase using molecular dynamic simulation and found out that only one surface loop (46~49) has flexible motion and this loop connects active sites pocket with second substrate binding site far from active site. However, flexible motion of this loop was constrained by cooperative interactions with other residues (cation-pi interaction (W42-R49) and electrostatic interaction (D4-R49)). This implies that flexible loop (46-49), especially, R49, seems to have important role in modulating stability and activity of xylanases. To validate this hypothesis, saturation mutagenesis of R49 was conducted and stability and kinetic parameters were determined. As expected, most mutants lost their stability due to disruption of cooperative interactions but gained higher catalytic power (up to 2 fold of Kcat /Km) due to increased flexibility. This explained typical trade-off between stability and activity of enzymes. Analysis on the flexible motion of enzyme using molecular modeling could be useful to identify important flexible residues involved in enzyme activity and stability. Further result and discussion will be presented.