Crystal Structure of the Pig Pancreatic α-Amylase Complexed with ρ-Nitrophenyl-α-D-Maltoside-Flexibility in the Active Site

Abstract

The X-ray structure analysis of a crystal of pig pancreatic α-amylase soaked with a ρ-nitrophenyl-α-D-maltoside (pNPG2) substrate showed a pattern of electron density corresponding to the binding of a ρ-nitrophenol unit at subsite −2 of the active site. Binding of the product to subsite −2 after hydrolysis of the pNPG2 molecules, may explain the low catalytic efficiency of the hydrolysis of pNPG2 by PPA. Except a small movement of the segment from residues 304–305 the typical conformational changes of the “flexible loop” (303–309), that constitutes the surface edge of the substrate binding cleft, were not observed in the present complex structure. This result supports the hypothesis that significant movement of the loop may depend on aglycone site being filled (Payan and Qian, J. Protein Chen. 22: 275, 2003). Structural analyses have shown that pancreatic α-amylases undergo an induced conformational change of the catalytic residue Asp300 upon substrate binding; in the present complex the catalytic residue is observed in its unliganded orientation. The results suggest that the induced reorientation is likely due to the presence of a sugar unit at subsite −1 and not linked to the closure of the flexible surface loop. The crystal structure was refined at 2.4 Å resolution to an R factor of 17.55% (R _free factor of 23.32%).