Role of glutamine-169 in the substrate recognition of human aminopeptidase B
Role of glutamine-169 in the substrate recognition of human aminopeptidase B
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谷氨酰胺-169 在人氨肽酶 B 底物识别中的作用
DOI：
10.1016/j.bbagen.2014.01.002
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Tsujimoto M
中科院分区：
文献类型：
--
作者：
Ogawa Y;Ohnishi A;Goto Y;Sakuma Y;Watanabe J;Hattori A;Tsujimoto M
氨基肽酶B（EC 3.4.11.6，AP B）优先水解合成底物和肽底物的N-末端碱性氨基酸。APB通过切割延伸的前体蛋白中的N-末端碱性氨基酸参与肽激素和神经递质如小胰高血糖素、胆囊收缩素和脑啡肽的产生和成熟。因此，我们认为，APB对碱性氨基酸的特异性是其生物学功能的关键。方法采用定点突变和S1位点的分子模拟技术，对人APB中负责碱性氨基酸偏好和酶促效率的氨基酸残基进行鉴定。结果Gln 169被Asn取代后，对荧光底物Lys-4-甲基香豆酰-7-酰胺的水解活性显著降低。MCA）。观察到对Arg-MCA的酶活性的实质性延迟，并且用Glu取代引起APB的酶活性的完全丧失。用Asn取代导致与APB的催化口袋相互作用的抑制剂的IC 50值增加。Asn突变体对氯离子结合的EC_（50）值也有所增加。Gln 169是肽底物最大裂解所需的。结论Gln 169是一种肽类激素和神经递质的加工酶，其催化口袋结构对获得最佳的酶活性和APB独特的碱性氨基酸选择性至关重要。
BackgroundAminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.MethodsSite-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.ResultsSubstitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50values of inhibitors that interact with the catalytic pocket of APB. The EC50value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.ConclusionGln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APBviamaintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.