1I45

YEAST TRIOSEPHOSPHATE ISOMERASE (MUTANT)

1I45 の概要

• エントリーDOI: 10.2210/pdb1i45/pdb
• 関連するPDBエントリー: 1YPI
• 分子名称: TRIOSEPHOSPHATE ISOMERASE (2 entities in total)
• 機能のキーワード: triosephosphate isomerase, mutant, yeast, 5'-fluorotryptophan, isomerase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 53566.68

構造登録者

Rozovsky, S.,Jogl, G.,Tong, L.,McDermott, A.E. (登録日: 2001-02-19, 公開日: 2001-06-30, 最終更新日: 2023-08-09)

主引用文献

Rozovsky, S.,Jogl, G.,Tong, L.,McDermott, A.E.
Solution-state NMR investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics.
J.Mol.Biol., 310:271-280, 2001

PubMed Abstract: Product release is partially rate determining in the isomerization reaction catalyzed by Triosephosphate Isomerase, the conversion of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, probably because an active-site loop movement is necessary to free the product from confinement in the active-site. The timescale of the catalytic loop motion and of ligand release were studied using 19F and 31P solution-state NMR. A 5'-fluorotryptophan was incorporated in the loop N-terminal hinge as a reporter of loop motion timescale. Crystallographic studies confirmed that the structure of the fluorinated enzyme is indistinguishable from the wild-type; the fluorine accepts a hydrogen bond from water and not from a protein residue, with minimal perturbation to the flexible loop stability. Two distinct loop conformations were observed by 19F NMR. Both for unligated (empty) and ligated enzyme samples a single species was detected, but the chemical shifts of these two distinct species differed by 1.2 ppm. For samples in the presence of subsaturating amounts of a substrate analogue, glycerol 3-phosphate, both NMR peaks were present, with broadened lineshapes at 0 degrees C. In contrast, a single NMR peak representing a rapid average of the two species was observed at 30 degrees C. We conclude that the rate of loop motion is less than 1400 s(-1) at 0 degrees C and more than 1400 s(-1) at 30 degrees C. Ligand release was studied under similar sample conditions, using 31P NMR of the phosphate group of the substrate analogue. The rate of ligand release is less than 1000 s(-1) at 0 degrees C and more than 1000 s(-1) at 30 degrees C. Therefore, loop motion and product release are probably concerted and likely to represent a rate limiting step for chemistry.

PubMed: 11419952
DOI: 10.1006/jmbi.2001.4673

実験手法

X-RAY DIFFRACTION (1.8 Å)