Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of beta-peptydyl aminopeptidase from Pseudomonas aeruginosa PAO1
Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of beta-peptydyl aminopeptidase from Pseudomonas aeruginosa PAO1
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活性位点丝氨酸残基与半胱氨酸的突变显示来自铜绿假单胞菌 PAO1 的 β-肽氨肽酶的酰基受体偏好的变化
DOI：
10.1007/s00253-013-4992-9
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发表时间：
2014
影响因子：
5
通讯作者：
Nobuhiro Mori
中科院分区：
文献类型：
--
作者：
Jiro Arima;Ayumi Tanaka;Masazumi Morimoto;Nobuhiro Mori
β-肽基氨基肽酶是属于P1家族的一种多肽酶，根据其水解性催化氨解反应。我们特异性地检测了铜绿假单胞菌PAO1的β-肽基氨基肽酶(BAPF)，以评估半胱氨酸或苏氨酸对催化丝氨酸突变对其催化能力的影响。重组BapF及其S237C突变体对β-高-甘氨酸-对-硝基苯胺(βhGly-PNA)具有释放活性，但酶反应产物完全不同。野生型BapF显示了βhGly-βhGly-PNA的合成活性，但产物在几分钟内消失，并转化为游离的βhGly。相反，S237C BapF可有效合成βhGly-βhGly-PNA而不被降解，这表明由于突变，该酶只识别氨基而不是水。此外，还观察到野生型和S237C BapF在酰基受体偏好上的差异。当以半胱胺为酰基受体时，βhGly-半胱胺只能在S237C BapF的作用下合成。相反，S237C BapF虽然是由野生型BapF合成的，但在以半胺为酰基受体的情况下不能合成βhGly-半胱胺。催化丝氨酸与半胱氨酸的突变导致酰基受体的这种动态变化被认为是P1家族多肽酶的一个独特特征。
A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase ofPseudomonas aeruginosaPAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibitedp-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.