Reaction mechanism of chalcone isomerase - pH dependence, diffusion control, and product binding differences
Reaction mechanism of chalcone isomerase - pH dependence, diffusion control, and product binding differences
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DOI：
10.1074/jbc.m109224200
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发表时间：
2002-01-11
影响因子：
4.8
通讯作者：
Noel, JP
中科院分区：
文献类型：
--
作者：
Jez, JM;Noel, JP
查耳酮异构酶（CHI）催化双环查耳酮分子内环化为三环（S）-黄烷酮。CHI的活性对于花色素和苯丙素类植物防御化合物的黄烷酮前体的生物合成是必需的。我们研究了4，2 ′，4 ′，6 ′-四羟基查耳酮、4，2 ′，4 ′-三羟基查耳酮、2 ′，4 ′-二羟基查耳酮和4，2 ′-二羟基查耳酮自发的和在CHI催化下的环化反应。黄烷酮形成的pH依赖性表明，非酶促和酶促反应首先需要底物2 '-羟基的体相电离，随后在C-环形成期间需要新形成的2'-含氧阴离子的反应性。溶剂粘度实验表明，在pH 7.5时，4，2 '，4'，6 '四羟基查耳酮、4，2'，4 '-三羟基查耳酮和2'，4 '-二羟基查耳酮的CHI催化的环化反应类似于90%扩散控制，而4，2'-二羟基查耳酮的环化受到可能反映2 '-羟基较高pK（α）的化学步骤的限制。在pH6.0时，与4，2 '，4'，6 '-四羟基查尔酮和4，2'，4 '-三羟基查尔酮的反应受50%扩散限制，而与二羟基查尔酮的反应受化学步骤限制。与产物7，4 ′-二羟基黄烷酮、7-羟基黄烷酮和4 ′-羟基黄烷酮复合的CHI的2.1-2.3埃分辨率晶体结构的比较表明，7-羟基黄烷酮都具有共同的结合模式，而4 ′-羟基黄烷酮在活性位点以改变的取向结合。我们的功能和结构的研究支持的建议，CHI加速立体化学定义的分子内环化成生物活性的（2S）-黄烷酮的选择性结合电离的查尔酮在构象有利于环闭合在扩散控制的反应。
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.