Xiang H, Dong J, Carey P R, Dunaway-Mariano D
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Biochemistry. 1999 Mar 30;38(13):4207-13. doi: 10.1021/bi982670b.
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 degrees 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-1 could 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.
天冬氨酸145在4-氯苯甲酰辅酶A脱卤酶的活性位点中起着至关重要的作用，在底物转化为4-羟基苯甲酰辅酶A的过程中，它在苯甲酸酯的4位形成一个瞬时共价键。用丙氨酸或丝氨酸等残基取代天冬氨酸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。前面的出版物（董等人）描述了其中一些复合物的拉曼光谱特征。本研究调查了由D145N脱卤酶与底物或产物形成的复合物。时间分辨吸收光谱和拉曼差光谱数据表明，这些体系随时间快速演变。对于底物复合物，最初吸收光谱和拉曼光谱显示底物结合在天冬酰胺145形式的酶活性位点中的特征，但这些特征伴随着离子化产物的特征。几分钟后，这些特征消失，取而代之的是与野生型脱卤酶活性位点中未离子化产物非常相似的特征。同样，对于产物复合物，吸收光谱和拉曼光谱最初显示D145N活性位点中离子化产物的证据，但这些很快被与结合到野生型酶上的未离子化产物非常相似的特征所取代。有人提出，结合在天冬酰胺145脱卤酶活性位点上的产物催化天冬酰胺侧链的脱酰胺作用，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酶群体中含有少量由自发脱酰胺作用形成的野生型酶，其产生产物。反过来，这些产物分子催化主要酶群体中天冬酰胺145的脱酰胺作用。因此，底物向产物的转化以及D145N向D145D脱卤酶的转化同时进行。通过让酶在pH 7.5的Hepes缓冲液中于室温下放置，对天冬酰胺145的自发脱酰胺作用进行了表征。在这些条件下，脱酰胺作用以0.0024 h-1的速率常数发生。通过停流动力学测量，在22℃的Hepes缓冲液中产物催化脱酰胺作用的速率为0.024 s-1，比自发过程快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附近的一个特征峰。这个谱带与底物或产物都无关，暂定为产物的4-O-基团攻击天冬酰胺侧链的羰基并随后释放氨形成的一种酯样物质。结合这些观察结果，提出了一个反应方案。
