6PTD

PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C MUTANT H32L

6PTD の概要

• エントリーDOI: 10.2210/pdb6ptd/pdb
• 分子名称: PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C (2 entities in total)
• 機能のキーワード: hydrolase, phosphoric diester, lipid degradation, phosphatidylinositol specific phospholipase c
• 由来する生物種: Bacillus cereus
• 細胞内の位置: Secreted: P14262
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 34543.68

構造登録者

Heinz, D.W. (登録日: 1997-07-18, 公開日: 1998-01-21, 最終更新日: 2024-05-22)

主引用文献

Gassler, C.S.,Ryan, M.,Liu, T.,Griffith, O.H.,Heinz, D.W.
Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis.
Biochemistry, 36:12802-12813, 1997

PubMed Abstract: The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.

PubMed: 9335537
DOI: 10.1021/bi971102d

実験手法

X-RAY DIFFRACTION (2.6 Å)