Cleavage of various peptides with pitrilysin from Escherichia coli:: Kinetic analyses using β-endorphin and its derivatives
Cleavage of various peptides with pitrilysin from Escherichia coli:: Kinetic analyses using β-endorphin and its derivatives
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DOI：
10.1271/bbb.68.2128
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发表时间：
2004-10-01
影响因子：
1.6
通讯作者：
Kanaya, S
中科院分区：
文献类型：
--
作者：
Cornista, J;Ikeuchi, S;Kanaya, S
以14个多肽为底物，对产自大肠杆菌的垂体溶酶进行了高效生产、纯化和酶活性分析。除了两个最小的多肽外，所有的肽都能在有限数量的位点上被垂体溶血素切割，但几乎没有显示出氨基酸的特异性。最有效地切割β-内啡肽(β-EP)，其K-m值为0.36um，k(CAT)值为750min(-1)。β-EP由31个残基组成，主要被Lys(19)-Asn(20)处的酶切割。用一系列N和/或C端截断和氨基酸取代的β-EP衍生物进行的动力学分析表明，β-EP中的三个疏水残基(Leu(14)、Val(15)和Leu(17))和区域22-26负责酶的高亲和力识别。这两个区域位于β-EP裂解位点的N-末端和C-末端，提示垂体菌素的底物结合口袋跨越其催化部位。
Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved beta-endorphin (beta-EP) most effectively, with a K-m value of 0.36 mum and a k(cat) value of 750 min(-1). beta-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys(19)-Asn(20). Kinetic analyses using a series of beta-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu(14), Val(15), and Leu(17)) and the region 22-26 in beta-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in beta-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.