117E

THE R78K AND D117E ACTIVE SITE VARIANTS OF SACCHAROMYCES CEREVISIAE SOLUBLE INORGANIC PYROPHOSPHATASE: STRUCTURAL STUDIES AND MECHANISTIC IMPLICATIONS

117E の概要

• エントリーDOI: 10.2210/pdb117e/pdb
• 分子名称: PROTEIN (INORGANIC PYROPHOSPHATASE), MANGANESE (II) ION, PHOSPHATE ION, ... (4 entities in total)
• 機能のキーワード: enzyme mechanism, iorganic pyrophosphatase, mutan structures, 2-metal ion mechanism, hydrolase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Cytoplasm: P00817
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 65203.21

構造登録者

Tuominen, V.,Heikinheimo, P.,Kajander, T.,Torkkel, T.,Hyytia, T.,Kapyla, J.,Lahti, R.,Cooperman, B.S.,Goldman, A. (登録日: 1998-09-15, 公開日: 1998-12-23, 最終更新日: 2023-08-09)

主引用文献

Tuominen, V.,Heikinheimo, P.,Kajander, T.,Torkkel, T.,Hyytia, T.,Kapyla, J.,Lahti, R.,Cooperman, B.S.,Goldman, A.
The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications.
J.Mol.Biol., 284:1565-1580, 1998

PubMed Abstract: We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 A and R-factors of 19.5% and 18.3%, respectively. In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile. The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site. These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the "two-metal ion" mechanism of polymerases.

PubMed: 9878371
DOI: 10.1006/jmbi.1998.2266

実験手法

X-RAY DIFFRACTION (2.15 Å)