Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.

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.

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 sp3-hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus.

Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ.

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

Carboxypeptidase B (EC 3.4.17.

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 Å and 1.62 Å to clarify the broad substrate specificity profile of the enzyme.

Molecular modeling of different substrate-binding modes and their role in penicillin acylase catalysis.

Molecular modeling was addressed to understand different substrate-binding modes and clarify the role of two positively charged residues of the penicillin G acylase active site - βR263 and αR145 - in binding of negatively charged substrates. Although the electrostatic contribution to productive substrate binding was dominated by βR263 rather than αR145, it was found that productive binding was not the only possible mode of substrate placement in the active site. Two extra binding modes - nonproductive and preproductive - were located by means of molecular docking and dynamics with binding affinities comparable with the productive one.