Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L-glutamic acid: reversed-polarity mutants.

  title={Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L-glutamic acid: reversed-polarity mutants.},
  author={Michael D. Edge and Cassie Forder and John F. Hennam and I Lee and David William Tonge and Ian M. Hardern and John Edward Fitton and Kay Eckersley and Simon J East and Ann J. Shufflebotham and Daniel Blakey and Alex Slater},
  journal={Protein engineering},
  volume={11 12},
Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues… Expand
Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues
The mechanism and structures of hCPO are disclosed, both ligand-free and -bound with a natural peptidic inhibitor ascribing the exquisite specificity toward C-t acidic residues to a single amino acid, Arg275, in the substrate-binding pocket, providing detailed information regarding determinants of enzyme specificity. Expand
Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.
3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond and the location/binding of SLys very closely resembled that of SArg. Expand
Structural principles of the broad substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T--role of amino acid residues at positions 260 and 262.
The obtained results confirm the important role of the amino acid residues at positions 260 and 262 in determination of the CPT substrate specificity. Expand
244 – Carboxypeptidase B
Publisher Summary This chapter describes the activity, specificity and structural chemistry of carboxypeptidase B. Carboxypeptidase B is highly specific for excising C-terminal Lys and Arg residuesExpand
Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine – a transition state analog of non-specific substrate
Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Expand
Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity
It is shown that the His68 residue is not a structural determinant of CPT specificity, and the removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64.7/1 to 162/1. Expand
Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity
The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate Specificity of the mutant enzyme. Expand
Caught after the Act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.
The structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, are provided, employing diffraction data to 1.6 A resolution. Expand
Isolation and Characteristics of Carboxypeptidase B from Zebra Blenny (Salaria basilisca) Viscera
Carboxypeptidase B from zebra blenny viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 28-fold increase in specific activity and 21.72% recovery. Expand
Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera
Carboxypeptidase B was purified from the pyloric ceca of the starfish, Asterina pectinifera and the N-terminal amino acid sequence of the enzyme was determined as ATFDYNKYHSYQEIMDWVTN. Expand


Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product, and all six antibodies recognize immunoblotted p62c- myc. Expand