Mechanism of chalcone synthase -: pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase
Mechanism of chalcone synthase -: pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase
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DOI：
10.1074/jbc.m008569200
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发表时间：
2000-12-15
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
聚酮合成酶(PHS)利用一种共同的机械策略组装结构多样的天然产物，这种策略依赖于半胱氨酸残基在一系列脱羧基缩合反应中锚定聚酮，从而形成最终反应产物。查尔酮合成酶(CHS)的结晶学和功能研究表明，半胱氨酸-组氨酸对(Cys(164)His(303))是催化机制的一部分。用硫醇特异性失活和丙二酰辅酶A脱羧基反应的pH依赖性来评估这两个残基之间的潜在相互作用。碘乙酰胺和碘乙酸对Cys(164)的灭活作用依赖于pH(pK(A)=5.50)。Cys(164)的酸性pK(A)表明，与His(303)的离子相互作用稳定了硫酸盐阴离子。与这一断言一致，用谷氨酰胺取代组氨酸(303)保持了催化活性，但使硫醇的pK(A)变为6.61。虽然H303A突变体是无催化活性的，但pH依赖的[C-14]碘乙酰胺掺入该突变体的pK(A)=7.62。随后分析了野生型CHS与H303Q和C164A催化的丙二酰辅酶A脱羧基反应的pH依赖性。突变体还支持在CHS活性部位存在离子对。半胱氨酸-组氨酸对在其他PKS的活性部位的结构和序列的保守性表明，硫代-咪唑离子对在聚酮的生物合成中起着核心作用。
Polyketide synthases (PHS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys(164)His(303)) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) maintains catalytic activity but shifts the pK(a) of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [C-14]iodoacetamide into this mutant exhibits a pK(a) = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A. mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.