1C9V

H12A VARIANT OF RIBONUCLEASE A

1C9V の概要

• エントリーDOI: 10.2210/pdb1c9v/pdb
• 関連するPDBエントリー: 1C8W 1C9X 3RSD 3RSK 3RSP 4RSD 4RSK
• 分子名称: RIBONUCLEASE A, CHLORIDE ION (3 entities in total)
• 機能のキーワード: antiparallel beta sheet, hydrolase
• 由来する生物種: Bos taurus (cattle)
• 細胞内の位置: Secreted: P61823
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 13712.16

構造登録者

Park, C.,Schultz, L.W.,Raines, R.T. (登録日: 1999-08-03, 公開日: 2001-06-27, 最終更新日: 2024-10-30)

主引用文献

Park, C.,Schultz, L.W.,Raines, R.T.
Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding.
Biochemistry, 40:4949-4956, 2001

PubMed Abstract: His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate complexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stability of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexation of wild-type RNase A and the variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, respectively. Determination of the stability of the complex of wild-type RNase A and 6-carboxyfluorescein approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the stability of the complex with 6-carboxyfluorescein approximately d(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic data provide the first thorough analysis of the contribution of histidine residues to nucleic acid binding.

PubMed: 11305910
DOI: 10.1021/bi0100182

実験手法

X-RAY DIFFRACTION (1.7 Å)