1RGK

RNASE T1 MUTANT GLU46GLN BINDS THE INHIBITORS 2'GMP AND 2'AMP AT THE 3' SUBSITE

1RGK の概要

• エントリーDOI: 10.2210/pdb1rgk/pdb
• 分子名称: RIBONUCLEASE T1, CALCIUM ION, ADENOSINE-2'-MONOPHOSPHATE, ... (4 entities in total)
• 機能のキーワード: hydrolase(endoribonuclease)
• 由来する生物種: Aspergillus oryzae
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 11481.01

構造登録者

Granzin, J.,Puras-Lutzke, R.,Landt, O.,Grunert, H.-P.,Heinemann, U.,Saenger, W.,Hahn, U. (登録日: 1992-02-19, 公開日: 1993-01-15, 最終更新日: 2024-10-23)

主引用文献

Granzin, J.,Puras-Lutzke, R.,Landt, O.,Grunert, H.P.,Heinemann, U.,Saenger, W.,Hahn, U.
RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.
J.Mol.Biol., 225:533-542, 1992

PubMed Abstract: On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.

PubMed: 1350642
DOI: 10.1016/0022-2836(92)90937-F

実験手法

X-RAY DIFFRACTION (1.87 Å)