title: [X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase].
abstract: The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 A resolution. The kinetic parameters of the hydrolysis of substrates of different lengths (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease in the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.
authors.forename: K M
authors.initials: KM
authors.lastname: Poliakov
authors.forename: D A
authors.initials: DA
authors.lastname: Goncharuk
authors.forename: A A
authors.initials: AA
authors.lastname: Trofimov
authors.forename: T N
authors.initials: TN
authors.lastname: Safonova
authors.forename: V A
authors.initials: VA
authors.lastname: Mit'kevich
authors.forename: E N
authors.initials: EN
authors.lastname: Tkach
authors.forename: A A
authors.initials: AA
authors.lastname: Makarov
authors.forename: A A
authors.initials: AA
authors.lastname: Shul'ga
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D012313:RNA; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D012313:RNA; D004722:Endoribonucleases; C022543:ribonuclease T(2)