The role of Glu-60 in the specificity of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(A) and RNA.
A computer model of the complex between G2'p5'G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2'p5'G. This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine. A barnase mutant was therefore made in which Glu-60 was replaced by Gln. This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat. For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined. The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A). This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A). For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair. Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km. In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate. The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding. The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations. Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters.
Bastyns
K
K
Laboratory of Chemical and Biological Dynamics, University of Leuven, Belgium.
Froeyer
M
M
Volckaert
G
G
Engelborghs
Y
Y
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D003198:Computer Simulation; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D009994:Osmolar Concentration; D011061:Poly A / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005971:Glutamates; D009841:Oligonucleotides; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease