Abstract
Carboxypeptidase T (CPT; EC 3.4.17.18) from Thermoactinomyces vulgaris is a distant homolog of the highly specific pancreatic carboxypeptidase B; but has a broad substrate specificity; the source of which remains unclear. A previous study of the structural bases of the substrate specificity of CPT using stable sulfamoyl analogs of the transition state of the elimination of leucine; phenylalanine; arginine; and glutamic acid; showed that the binding of the C-terminal residue of the substrate to the primary selectivity pocket of CPT leads to a change in the distance between Zn2+ and the sulfur atom. This value is related to the efficiency of catalysis of the corresponding substrate or the inhibition constant of the corresponding stable analog of the transition state. In this work; we obtained crystallographic and kinetic data of the complex of CPT with N-sulfamoyl-L-valine; confirming the effect of the binding of the ligand’s side group by the primary specificity pocket of CPT on the structure of the catalytic center; which can explain the unusual substrate specificity of CPT.
Highlights
IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
Experiments final model of the Carboxypeptidase T (CPT)-SVal complex was refined to a resolution of 1.92 Å
The CPT polypeptide chain was complete and clearly traced on the electron density map (Asp1-Cys323), the active center of the enzyme contained electron density, which was interpreted as SVal molecule (Figure S1)
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Increasing attention to them is caused by their involvement in important physiological processes such as food digestion, neuropeptide processing, blood clotting, inflammation, and carcinogenesis [1,2,3]. MCPs are used in biotechnology such as insulin production. In this regard, there is an interest in the rational redesign of MCPs to improve their activity, thermal stability, and substrate specificity. The successful enzyme design is based on a correct understanding of the substrate recognition mechanism
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