The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

Abstract

The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp 3 -hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of PLOS ONE | https://doi.

Study of the Interaction of Sorption and Catalytic Centers in Carboxypeptidase T by X-ray Analysis

“…The sulfamoyl part of SVal had a tetrahedral configuration that simulated sp 3 -hybridized C-atom configuration in a complex with the substrate. The sulfamoyl binding mode was similar to those found in previously reported CPA-SPhe (PDB ID: 1IY7 [32] and CPT-SLeu (PDB ID: 6GO2 [16]) complexes. The SVal side chain was anchored in the S1 -subsite, which is the pocket of the primary substrate specificity of the CPT.…”

“…We have previously shown [16] that the geometry of the transition complex analog formed upon binding of the sulfamoyl transition state analog to the active center of CPT depends on the nature of the side group of the sulfamoyl inhibitor. It is expressed in the fact that the length of the connections between the ligand atoms and the catalytic center groups in enzyme complexes changes as one ligand switched for another.…”

“…Inhibitors' binding constants were determined using the integral kinetics method. Progress curves of the specific substrate N-(benzyloxycarbonyl)-Ala-Ala-Leu-OH (ZAAL) conversion were registered in the presence of different concentrations of inhibitor and then linearized as described earlier [16] to obtain apparent KM over Vmax values (KMapp/Vmax). Then "inhibitor concentration-KMapp/Vmax" data points were later fitted to the linear equations y = ax + b, from which inhibition constants were calculated as b/a.…”

“…In the case of CPT, the position of the substrate's side chain in the enzyme-substrate complex may change (V. Akparov et al, 2015). The structure of the tetrahedral transition complex also changes from one substrate to another [16]. In this case, the recognition of the substrate is carried out according to the principle of induced fit.…”