Role of histidine 124 in the catalytic function of ribonuclease HI from Escherichia coli.
The role of the conserved histidine residue (His124) in the catalytic function of Escherichia coli ribonuclease HI was probed by the use of the pH titration experiments with 1H NMR and site-directed mutagenesis. His124 is located close to the catalytic triad formed by the three carboxylates. The C2H proton resonance of His124, as well as those of four other His residues, in the enzyme were assigned by comparing the 1H NMR spectrum of the wild-type enzyme to the spectra of the five mutant enzymes, each of which lacked a different His residue. From the analysis of the pH titration shifts of these resonances, the pK alpha values of all His residues were determined. The pK alpha value of His124 was 7.1, which is slightly higher than, but close to, the normal value of the imidazole group. This result suggests that His124 is located in an acidic environment but does not interact directly with a carboxyl group in the solution structure. Three mutant enzymes, in which His124 was replaced by Lys, Gln, or Glu were constructed. The kinetic parameters of these mutant enzymes were determined by using the M13 DNA/RNA hybrid as a substrate. Substitution of Lys, Gln, or Glu for His124 dramatically lowered the Vmax value by 30-100-fold, without significantly affecting the Km value, as compared to those of the wild-type enzyme. In addition, any mutation impaired the in vivo function of the enzyme. These results indicate that His124 is involved in the catalytic function of the enzyme. We propose that His124 changes its conformation in the catalytic reaction and enhances the catalytic efficiency by removing a proton from a catalytically essential carboxylate.
Protein Engineering Research Institute, Osaka, Japan.
Y
Y
Oda
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M
Yoshida
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S
Kanaya
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006639:Histidine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*
D006639:Histidine; D016914:Ribonuclease H; C078856:ribonuclease HI