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首页> 外文期刊>Biochemistry >Role of Active Site Binding Interactions in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis
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Role of Active Site Binding Interactions in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis

机译：活性位点结合相互作用在4-氯苯甲酰基辅酶A脱卤酶催化中的作用

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摘要

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehhalogenase catalyzes the hydrolytic dehalogenatin of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. this study examines the role of binding interactins in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-Coa complex. these groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interactions. The chanrges in the Gibbs free energy of the enzyme-substrate complex (#DELTA##DELTA#G_(ES))and enzyme-transition state cmplex ((#DELTA##DELTA#G) brought about by the modification were measured. Cases where (#DELTA##DELTA#G exceeds (#DELTA##DELTA#G_(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis we show that the H-bond interactions between the Cly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducting the energy barrier to catalysis by an additional 3,3 kcal/mol. together, these binding interactions contribute approx10~6 to the k_(cat)/K_m.
机译：4-氯苯甲酰基辅酶A（4-CBA-CoA）脱卤素酶通过涉及Asp145初始攻击C（4）的多步机制催化4-CBA-CoA水解为4-羟基苯甲酰基-CoA（4-HBA-CoA）底物的苯甲酰基环上形成一个Meisenheimer中间体（EMc），然后排出氯离子以形成一个芳基化的酶中间体（EAr），然后在EAr中进行酯水解形成产物。这项研究探讨了结合相互作用素在脱卤酶催化中的作用。从酶-4-HBA-3'-脱磷-Coa复合物的X射线晶体结构中鉴定出定位成有利于结合相互作用的酶和底物基团。为了破坏结合相互作用，对这些基团分别进行了修饰（通过定点诱变或化学合成）。测量了修饰引起的酶-底物复合物（＃DELTA ## DELTA＃G_（ES））和酶-过渡态复合物（（＃DELTA ## DELTA＃G））的吉布斯自由能的变化。其中（＃DELTA ## DELTA＃G超过（＃DELTA ## DELTA＃G_（ES）表示用于催化的结合相互作用。基于此分析，我们表明Cly114与Phe64主链之间的H键相互作用酰胺NHs和底物苯甲酰基C = O基团在限速过渡态上额外提供了3.1 kcal / mol的稳定度，酶和底物CoA核苷酸部分之间的结合相互作用在限速过渡态中也增强了，还原通过额外的3.3 kcal / mol的能量屏障，这些结合相互作用对k_（cat）/ K_m贡献约10〜6。

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