Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-arginine.

Porcine pancreatic carboxypeptidase B (EC 3.4.23.6) was complexed with a stable transition-state analogue, N-sulfamoyl-L-arginine, in which an S atom imitates the sp(3)-hybridized carbon in the scissile-bond surrogate. Crystals were grown in a form belonging to the same space group, P41212, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 Å. The molecule was refined and the positions of non-H atoms of the inhibitor and water molecules were defined using difference Fourier maps. The enzyme-inhibitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation between the complexed and uncomplexed forms of carboxypeptidase B are shown. The inhibitor is bound in a curved conformation in the active-site cleft, and the sulfamide group is bound to the Zn ion in an asymmetric bidentate fashion. The complex is stabilized by hydrogen bonds between the N1/N2 guanidine group of the inhibitor and the Asp255 carboxyl of the enzyme. The side-chain CH2 groups of the inhibitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may bind to carboxypeptidase B and therefore provides an insight into the structural basis of carboxypeptidase B selectivity, which is useful for the rational design of a carboxypeptidase with improved selectivity for industrial recombinant pro-insulin processing.