Reaction mechanism of chalcone isomerase - pH dependence, diffusion control, and product binding differences

被引:127
作者
Jez, JM [1 ]
Noel, JP [1 ]
机构
[1] Salk Inst Biol Studies, Struct Biol Lab, La Jolla, CA 92037 USA
关键词
D O I
10.1074/jbc.M109224200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)- flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are similar to90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(alpha) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are similar to50% diffusion-limited, whereas the reactions of both dihy-droxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 Angstrom resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction.
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页码:1361 / 1369
页数:9
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