Three-Dimensional Structure of a Mutant of Carboxypeptidase T from Thermoactinomyces vulgaris Bearing an Implanted S1' Subsite of Pancreatic Carboxypeptidase B Complexed with a Product Analog

Abstract

A mutant of bacterial carboxypeptidase T from Thermoactinomyces vulgaris (CPT5) with amino-acid substitutions in the S1' specificity pocket of the active site for the residues corresponding to the S1' region of pancreatic carboxypeptidase B (Gly215Ser, Ala251Gly, Thr257Ala, Asp260Gly, Thr262Asp) was crystallized in complex with N-BOC-L-Leu. The latter occupies the S1 subsite of the active site and is a reaction product analog. The X-ray diffraction data set was collected from a crystal of CPT5 at the SPring 8 synchrotron facility. The three-dimensional structure of CPT5 was determined at 1.40 Å resolution and refined to Rfact = 13.6%, Rfree = 12.5%. The binding of N-BOC-L-Leu to the S1 subsite of CPT5 leads to conformational changes in the protein concerning primarily the Сα atoms of five residues of the flexible loop at the interface between the S1 and S1' subsites and the side chains of the residues Tyr255 and Leu254 involved in the induced fit. Besides, conformational changes are observed in the active-site residues Glu277 and Asp262 and the side chains of residues located on the surface of the protein molecule. The observed changes differ from the conformational changes, which occur upon binding of N-BOC-L-Leu to wild-type CPT. This fact is indicative of the mutual influence of the S1 and S1' subsites in metallocarboxypeptidase T.