Identification of active site residues essential to 4-chlorobenzoyl-coenzyme A dehalogenase catalysis by chemical modification and site directed mutagenesis.
Identification of active site residues essential to 4-chlorobenzoyl-coenzyme A dehalogenase catalysis by chemical modification and site directed mutagenesis.
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DOI：
10.1021/bi9609533
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发表时间：
1996-08
期刊：
影响因子：
2.9
通讯作者：
Guang Yang;Rui-Qin Liu;K. Taylor;Hong Xiang;John C. Price;D. Dunaway-Mariano
中科院分区：
文献类型：
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作者：
Guang Yang;Rui-Qin Liu;K. Taylor;Hong Xiang;John C. Price;D. Dunaway-Mariano
4-氯苯甲酰基辅酶A（4-CBA-CoA）脱卤酶通过亲核芳族取代途径催化4-CBA-CoA水解为4-羟基苯甲酰基辅酶A（4-HBA-CoA），该途径涉及活性位点羧酸酯侧链参与共价催化。在本文中，我们报告的保守的天冬氨酸，组氨酸和色氨酸残基的鉴定4-CBA-CoA催化使用化学修饰和定点突变技术。用焦碳酸二乙酯处理脱卤酶导致催化活性完全丧失（Kinact = 0.17 mM-1 min-1，pH 6.5，25 ℃），其通过随后用羟胺处理完全恢复。由酶结合的4-HBA-CoA提供的失活保护表明必需的组氨酸残基位于活性位点。保守的组氨酸残基81，90，94，和208与谷氨酰胺残基的取代导致的催化活性的显着损失，只有在组氨酸81和90突变体的情况下。底物和产物配体结合研究表明，结合在这些突变体中没有显著抑制。选择保守的天冬氨酸和谷氨酸残基的定点诱变，确定天冬氨酸145是脱卤酶催化所必需的。配体结合研究表明，该残基不需要紧密的底物/产物结合。用N-溴代琥珀酰亚胺对脱卤酶进行化学修饰导致催化活性完全丧失，这是通过用产物配体饱和活性位点来防止的，提供了有利于必需活性位点色氨酸的证据。苯丙氨酸取代保守的色氨酸残基179和137只在后者（Kcat = 0.03%的野生型脱卤酶）的催化活性降低。基于这些结果和最近测定的4-CBA-CoA脱卤酶和4-HBA-CoA的复合物的X射线晶体结构[Benning，M. M.，泰勒，K.L.，柳河，巴西-地问：杨，G.，Xiang，H.，中国农业科学院，Wesenberg，G.，Dunaway-Mariano，D.，霍尔顿（1996）Biochemistry 35，8103 -8109]我们提出天冬氨酸145起活性位点亲核试剂的作用，色氨酸137起天冬氨酸145 C = O的氢键供体的作用，组氨酸90起使结合的H2O分子去质子化的作用。
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) via a nucleophilic aromatic substitution pathway involving the participation of an active site carboxylate side chain in covalent catalysis. In this paper we report on the identification of conserved aspartate, histidine, and tryptophan residues essential to 4-CBA-CoA catalysis using chemical modification and site-directed mutagenesis techniques. Treatment of the dehalogenase with diethyl pyrocarbonate resulted in complete loss of catalytic activity (Kinact = 0.17 mM-1 min-1 at pH 6.5, 25 degrees C) that was fully regained by subsequent treatment with hydroxylamine. The protection from inactivation afforded by enzyme bound 4-HBA-CoA indicated that the essential histidine residues are located at the active site. Replacement of conserved histidine residues 81, 90, 94, and 208 with glutamine residues resulted in a significant loss of catalytic activity only in the cases of the histidine 81 and 90 mutants. Substrate and product ligand binding studies showed that binding is not significantly inhibited in these mutants. Site directed mutagenesis of a selection of conserved aspartate and glutamate residues, identified aspartate 145 as being essential to dehalogenase catalysis. Ligand binding studies showed that this residue is not required for tight substrate/product binding. Chemical modification of the dehalogenase with N-bromosuccinimide resulted in full loss of catalytic activity that was prevented by saturation of the active site with product ligand, providing evidence favoring an essential active site tryptophan. Phenylalanine replacement of conserved tryptophan residues 179 and 137 reduced catalytic activity only in the latter (Kcat = 0.03% of wild-type dehalogenase). On the basis of these results and the recently determined X-ray crystal structure of the complex of 4-CBA-CoA dehalogenase and 4-HBA-CoA [Benning, M. M., Taylor, K.L., Liu, R.-Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D., Holden, H.M. (1996) Biochemistry 35,8103-8109] we propose that aspartate 145 functions as the active site nucleophile, that tryptophan 137 serves as a hydrogen bond donor to the aspartate 145 C = O, and that histidine 90 serves to deprotonate the bound H2O molecule.