title: Replacement of His12 or His119 of bovine pancreatic ribonuclease A with acidic amino acid residues for the modification of activity and stability.
abstract: In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K(m) values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K(m): the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A. Alkylation with iodoacetic acid (IAA) revealed that mutant enzymes had reduced reaction rates and that no modification was evident at Glu12 and Asp12 of H12E and H12D, respectively. This indicates that the low catalytic activity of mutant enzymes could be due to low basicity of Asp12 and Glu12. While the T(m) of H119D was almost the same as that of the wild-type enzyme, the T(m) of both H12D and H12E markedly decreased. It became apparent that His12 located at the bottom of the active site cleft contributes significantly to the structural stability of RNase A.
authors.affiliation: Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, The Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanimizu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueno
authors.forename: Rikimaru
authors.initials: R
authors.lastname: Hayashi