Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis.
The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.
Haruki
M
M
Protein Engineering Research Institute, Osaka, Japan.
Noguchi
E
E
Nakai
C
C
Liu
Y Y
YY
Oobatake
M
M
Itaya
M
M
Kanaya
S
S
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005798:Genes, Bacterial; D006639:Histidine; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D013489:Suppression, Genetic; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D001216:Asparagine; D016914:Ribonuclease H; C078856:ribonuclease HI