Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes
Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes
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DOI：
10.1073/pnas.94.22.11986
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发表时间：
1997-10-28
影响因子：
11.1
通讯作者：
Shimizu, S
中科院分区：
文献类型：
--
作者：
Kobayashi, M;Fujiwara, Y;Shimizu, S
主要是基于以前进行的各种抑制剂研究，酰胺酶被认为是巯基酶，但并不完全满意这种普遍接受的解释，我们对紫红红球菌J1的一种特殊酰胺酶进行了一系列定点诱变研究，该酶参与其腈代谢。重组酰胺酶在大肠杆菌中作为包涵体产生，以极大地促进其回收和随后的纯化。关于推定的活性位点残基Cys 203，Cys 203 → Ala突变体酶的比活仍保持原来的11.5%，与此形成鲜明对比的是，在Cys 203附近的某些其它位置的取代对酰胺酶的影响要大得多，Asp 191的谷氨酸取代使突变体酶的比活降低到野生型酶活的1.33%，此外，Asp 191-> Bsn取代以及Ser 195-> Ala取代完全消除了比活性。因此看来，在所有酰胺酶共有的所谓特征序列中存在的各种保守残基中，真实的活性位点残基是Asp 191和Ser 195而不是Cys 203。由于酰胺底物中的酰胺键（CO-NH 2）在结构上与肽键（CO-NH-）没有太远的距离，因此将各种酰胺酶的特征序列与各种类型的蛋白酶的活性位点序列进行比较。结果发现，天冬氨酸和丝氨酸残基对应的Asp 191和Ser 195的红球菌酰胺酶的天冬氨酸蛋白酶的活性位点序列内存在，从而表明两者之间的进化关系。
Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes, Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism, For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification, With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity, In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase, Glutamic acid substitution of Asp191 reduced the specific activity of the mutant Enzyme to 1.33% of the wild-type activity, Furthermore, Asp191 --> Bsn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity, It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.