Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme.
Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme.
复制标题
产品催化 D145N 脱卤素酶的脱酰胺作用，产生野生型酶。
DOI：
10.1021/bi982670b
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Dunaway-Mariano,D
中科院分区：
文献类型：
--
作者：
Xiang,H;Dong,J;Carey,PR;Dunaway-Mariano,D
天冬氨酸145在4-氯苯甲酰基-CoA脱卤酶的活性位点中起重要作用，在底物转化为4-羟基苯甲酰基-CoA期间在苯甲酸酯的4位形成瞬时共价键。用丙氨酸或丝氨酸等残基取代Asp 145会导致完全失活，并且可以与底物或产物形成稳定的复合物。后者中的一些的拉曼光谱表征描述于先前的出版物（Dong等人）中。本工作研究D145 N脱卤酶和底物或产物形成的复合物。时间分辨吸收和拉曼差光谱数据表明，这些系统随时间迅速演变。对于底物复合物，最初的吸收和拉曼光谱显示结合在酶的天冬酰胺145形式的活性位点中的底物的特征，但是这些特征伴随着电离产物的特征。几分钟后，这些特征消失，被野生型脱卤酶活性位点中与未电离产物非常相似的特征所取代。类似地，对于产物复合物，吸收和拉曼光谱最初显示D145 N的活性位点中的离子化产物的证据，但是这些被与结合至野生型酶的未离子化产物非常相似的特征迅速取代。提出与天冬酰胺145脱卤酶的活性位点结合的产物催化天冬酰胺侧链的脱酰胺化以产生野生型天冬氨酸145。对于涉及底物的复合物，天冬酰胺145酶群含有少量WT酶，其通过自发脱酰胺形成，产生产物。反过来，这些产物分子催化主要酶群中Asn 145的脱酰胺作用。因此，底物向产物的转化和D145 N向D145 D脱卤酶的转化同时进行。天冬酰胺145的自发脱酰胺化的特征在于使酶在室温下在pH 7.5的Hepes缓冲液中静置。在这些条件下发生脱酰胺反应，速率常数为0.0024 h-1。通过停流动力学测量，在22 °C下在Hepes缓冲液中产物催化的脱酰胺的速率比自发过程快0.024 s-1，36000倍。在底物和产物-酶复合物的早期拉曼光谱中可以观察到1570 cm-1附近的特征。该条带与底物或产物均不相关，并且暂时归属于通过产物的4-O-基团攻击天冬酰胺侧链的羰基并随后释放氨而形成的酯样物质。提出了一个反应方案，将这些意见。
Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 °C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1could be observed in the early Raman spectra of both substrate and product−enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O-group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.