Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate.
To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1. 9-fold. In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124). These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.
Haruki
M
M
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan. haruki@ap.chem.eng.osaka-u.ac.jp
Tsunaka
Y
Y
Morikawa
M
M
Iwai
S
S
Kanaya
S
S
D000409:Alanine / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism
D009841:Oligonucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D013873:Thionucleotides; D006639:Histidine; D016914:Ribonuclease H; C078856:ribonuclease HI; D000409:Alanine