title: Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.
abstract: Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) 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. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.
authors.affiliation: a Protein Chemistry Department , State Research Institute for Genetics and Selection of Industrial Microorganisms , 1-yi DorozhnyiProezd 1, Moscow , 117545 , Russia.
authors.forename: Valery
authors.initials: V
authors.lastname: Akparov
authors.affiliation: b X-ray Analysis Methods and Synchrotron Radiation Laboratory , Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences , Leninskii Prospect 59, Moscow , 119333 , Russia.|c NBICS Center, National Research Centre 'Kurchatov Institute' , Akad. Kurchatov Sq. 1, Moscow , 123182 , Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Timofeev
authors.affiliation: d Laboratory of Molecular Genetics, Moscow Institute of Physics and Technology (State University) , 9 Institutsky per. Dolgoprudny, Moscow , 141700 , Russia.
authors.forename: Ilyas
authors.initials: I
authors.lastname: Khaliullin
authors.affiliation: e Belozersky Institute of Physicochemical Biology , Lomonosov Moscow State University , 1/40Leninskie Gory, Moscow , 119991 , Russia.
authors.forename: Vytas
authors.initials: V
authors.lastname: Švedas
authors.affiliation: b X-ray Analysis Methods and Synchrotron Radiation Laboratory , Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences , Leninskii Prospect 59, Moscow , 119333 , Russia.|c NBICS Center, National Research Centre 'Kurchatov Institute' , Akad. Kurchatov Sq. 1, Moscow , 123182 , Russia.
authors.forename: Inna
authors.initials: I
authors.lastname: Kuranova
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D043424:Carboxypeptidase B / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D010649:Phenylalanine; D043424:Carboxypeptidase B
keywords: N-sulfamoyl-L-Phenylalanine; X-ray analysis; carboxypeptidase B; substrate selectivity determinants; transition state complex