1ELY

E. COLI ALKALINE PHOSPHATASE MUTANT (S102C)

1ELY の概要

• エントリーDOI: 10.2210/pdb1ely/pdb
• 分子名称: ALKALINE PHOSPHATASE, ZINC ION, MAGNESIUM ION, ... (5 entities in total)
• 機能のキーワード: alkaline phosphatase, hydrolase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Periplasm: P00634
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 94721.11

構造登録者

Stec, B.,Hehir, M.,Brennan, C.,Nolte, M.,Kantrowitz, E.R. (登録日: 1998-02-10, 公開日: 1998-05-27, 最終更新日: 2024-10-16)

主引用文献

Stec, B.,Hehir, M.J.,Brennan, C.,Nolte, M.,Kantrowitz, E.R.
Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.
J.Mol.Biol., 277:647-662, 1998

PubMed Abstract: Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

PubMed: 9533886
DOI: 10.1006/jmbi.1998.1635

実験手法

X-RAY DIFFRACTION (2.8 Å)