Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.
All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.
Oda
Y
Y
Protein Engineering Research Institute, Osaka, Japan.
Yamazaki
T
T
Nagayama
K
K
Kanaya
S
S
Kuroda
Y
Y
Nakamura
H
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016914:Ribonuclease H / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium