A NEW, SENSITIVE FLUOROGENIC SUBSTRATE FOR PAPAIN BASED ON THE SEQUENCE OF THE CYSTATIN INHIBITORY SITE
A NEW, SENSITIVE FLUOROGENIC SUBSTRATE FOR PAPAIN BASED ON THE SEQUENCE OF THE CYSTATIN INHIBITORY SITE
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DOI：
10.1006/abbi.1993.1516
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发表时间：
1993-11-01
影响因子：
3.9
通讯作者：
JULIANO, L
中科院分区：
文献类型：
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作者：
GAUTHIER, F;MOREAU, T;JULIANO, L
我们设计并测试了一种具有分子内猝灭荧光的新型木瓜蛋白酶底物。它是基于参与半胱氨酸蛋白酶抑制的胱抑素超家族所有成员的高度保守的序列。该底物O-氨基苯甲酰基(Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl(EDDnp)对木瓜酶非常敏感，其二级反应速率常数kcat/Km值为3.1×107M−1s−1。该底物也能被组织蛋白酶L有效地降解，尽管该酶的Kcat/Km值比木瓜酶低约60倍。这种变化是由于油墨的减少，Km的几乎是一样的。这使得这两种酶之间有明显的功能区别，并可能导致针对个别半胱氨酸蛋白酶的选择性抑制剂的开发。与最常用的木瓜酶底物不同，ABZ-QVVAGA-EDDnp不被胰酶水解。N-末端氨基酸序列分析表明，木瓜酶的裂解位点为A-G键。使用敏感和特定的底物，例如这里描述的一种，将被证明对于研究寄生虫感染中的半胱氨酸蛋白酶活性是非常有价值的。比较了木瓜蛋白酶或组织蛋白酶I与Abz-QVVAGA-EDDnp的紧密相互作用与与胱抑素抑制剂的密切相互作用，后者的结构中都包含一个QxVxG共识片段。
We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-arninobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate constantkcat/Kmof 3.1 107M−1s−1. It is also efficiently hydrolyzed by cathepsin L, although thekcat/Kmfor this proteinase is about 60-fold lower than that for papain. This change is due to a decrease inkcat, theKm′s are almost identical. This allows clear functional discrimination between these two proteinases, and may lead to the development of selective inhibitors for individual cysteine proteinases. Unlike most commonly used papain substrates, Abz-QVVAGA-EDDnp is not hydrolyzed by trypsin. The papain cleavage site was identified as the A-G bond by N-terminal amino acid sequencing. The use of sensitive and specific substrates such as the one described here will prove invaluable for investigating cysteine proteinase activities in parasite infections. The close interaction between papain or cathepsin I with Abz-QVVAGA-EDDnp is compared to that with cystatin inhibitors, which all include a QxVxG consensus segment in their structure.