1JX0

Chalcone Isomerase--Y106F mutant

1JX0 の概要

• エントリーDOI: 10.2210/pdb1jx0/pdb
• 関連するPDBエントリー: 1EYP 1JX1
• 分子名称: CHALCONE--FLAVONONE ISOMERASE 1, SULFATE ION, 7-HYDROXY-2-(4-HYDROXY-PHENYL)-CHROMAN-4-ONE, ... (4 entities in total)
• 機能のキーワード: monomer, unique fold, isomerase
• 由来する生物種: Medicago sativa
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 48310.97

構造登録者

Jez, J.M.,Bowman, M.E.,Noel, J.P. (登録日: 2001-09-05, 公開日: 2002-07-24, 最終更新日: 2024-04-03)

主引用文献

Jez, J.M.,Bowman, M.E.,Noel, J.P.
Role of Hydrogen Bonds in the Reaction Mechanism of Chalcone Isomerase
Biochemistry, 41:5168-5176, 2002

PubMed Abstract: In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.

PubMed: 11955065
DOI: 10.1021/bi0255266

実験手法

X-RAY DIFFRACTION (2.85 Å)