Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr607 residue
Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr607 residue
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DOI：
10.1016/j.abb.2010.05.025
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发表时间：
2010-08-15
影响因子：
3.9
通讯作者：
Carmona, Adriana K.
中科院分区：
文献类型：
--
作者：
Lorenzon, Ricardo Z.;Cunha, Carlos E. L.;Carmona, Adriana K.
寡肽酶A（Oligopeptidase A，OpdA）属于细菌肽酶的M3 A亚家族，具有与哺乳动物硫代寡肽酶（thimet-oligopeptidase，TOP）和神经溶解素（neurolysin，NEL）类似的催化和结构特性。这三种酶在靠近催化位点的柔性环上具有四个保守的Tyr残基。在OpdA中，柔性环由残基600-614（（600）SHIFAGGYAAGYYSY（614））形成。模拟研究表明，在OpdA中，Tyr（607）残基可能参与底物的识别，在催化中起关键作用。用Phe（Y 607 F）或Ala（Y 607 A）取代Tyr（607），构建了两个突变体，并考察了定点突变对催化过程的影响。Abz-GXSPFRQ-EDDnp衍生物的水解（Abz =邻氨基苯甲酸; EDDnp N-[2，4-二硝基苯基]-乙二胺; X =不同的氨基酸）以比较野生型OpdA（OpdA WT）与Y 607 F和Y 607 A突变体的活性。而Y 607 F和Y 607 A突变体能够水解X-S和P-F键。动力学参数显示Tyr（607）在OpdA催化活性中的重要性，因为其取代促进k（cat）/K-m值的降低，对于Y 607 F突变体降低约100倍，对于Y 607 A降低约1000倍。然而，这两种突变并不影响蛋白质折叠所示的CD和内在荧光分析。我们的研究结果表明，OpdA Tyr（607）残基在酶-底物相互作用和水解活性中起着重要作用。(C)2010年爱思唯尔公司All rights reserved.
Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 ((600)SHIFAGGYAAGYYSY(614)). Modeling studies indicated that in OpdA the Tyr(607) residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr(607) by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr(607) in OpdA catalytic activity as its substitution promoted a decrease in the k(cat)/K-m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr(607) residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity. (C) 2010 Elsevier Inc. All rights reserved.