1MCZ

BENZOYLFORMATE DECARBOXYLASE FROM PSEUDOMONAS PUTIDA COMPLEXED WITH AN INHIBITOR, R-MANDELATE

1MCZ の概要

• エントリーDOI: 10.2210/pdb1mcz/pdb
• 関連するPDBエントリー: 1BFD
• 分子名称: BENZOYLFORMATE DECARBOXYLASE, MAGNESIUM ION, THIAMINE DIPHOSPHATE, ... (5 entities in total)
• 機能のキーワード: decarboxylase, thiamin diphosphate, r-mandelate, lyase
• 由来する生物種: Pseudomonas putida
• タンパク質・核酸の鎖数: 16
• 化学式量合計: 912266.63

構造登録者

Polovnikova, E.S.,Bera, A.K.,Hasson, M.S. (登録日: 2002-08-06, 公開日: 2003-02-25, 最終更新日: 2024-02-14)

主引用文献

POLOVNIKOVA, E.S.,McLeish, M.J.,Sergienko, E.A.,Burgner, J.T.,Anderson, N.L.,BERA, A.K.,Jordan, F.,Kenyon, G.L.,HASSON, M.S.
Structural and Kinetic Analysis of Catalysis by a Thiamin Diphosphate-Dependent Enzyme, Benzoylformate Decarboxylase
Biochemistry, 42:1820-1830, 2003

PubMed Abstract: Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.

PubMed: 12590569
DOI: 10.1021/bi026490k

実験手法

X-RAY DIFFRACTION (2.8 Å)