title: Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant.
abstract: Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russian Federation.
authors.forename: V A
authors.initials: VA
authors.lastname: Mitkevich
authors.forename: A A
authors.initials: AA
authors.lastname: Schulga
authors.forename: A A
authors.initials: AA
authors.lastname: Trofimov
authors.forename: P V
authors.initials: PV
authors.lastname: Dorovatovskii
authors.forename: D A
authors.initials: DA
authors.lastname: Goncharuk
authors.forename: E N
authors.initials: EN
authors.lastname: Tkach
authors.forename: A A
authors.initials: AA
authors.lastname: Makarov
authors.forename: K M
authors.initials: KM
authors.lastname: Polyakov
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011069:Poly I / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D050505:Mutant Proteins; D011069:Poly I; D004722:Endoribonucleases; C022543:ribonuclease T(2)
keywords: binase; cytotoxicity; enzymatic activity; ribonucleases; site-directed mutagenesis