Titration and mapping of the active site of cysteine proteinases from Porphyromonas gingivalis (Gingipains) using peptidyl chloromethanes
Titration and mapping of the active site of cysteine proteinases from Porphyromonas gingivalis (Gingipains) using peptidyl chloromethanes
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DOI：
10.1515/bchm.1997.378.3-4.223
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发表时间：
1997-03-01
影响因子：
3.7
通讯作者：
Travis, J
中科院分区：
文献类型：
--
作者：
Potempa, J;Pike, R;Travis, J
牙龈卟啉单胞菌是与牙周疾病相关的主要病原体之一，它释放被称为牙龈痛的强大的半胱氨酸蛋白酶，这是该细菌的关键毒力因子。在精氨酸(R)或赖氨酸(K)残基之后特异裂解的三种形式的牙周疼痛，即牙周疼痛R1、牙周疼痛R2(牙周疼痛Rs)和牙周疼痛K，根据它们与一系列合成肽基氯甲烷抑制剂和多肽(酰氧基)甲烷相互作用的动力学特征进行了表征。氯甲烷抑制剂对所有酶都有不同程度的抑制作用，这取决于抑制剂的多肽成分。因此，在P1处含有碱性残基的抑制剂可以迅速使牙周疼痛失活，并且在P2处可以看到一些特异性。(酰氧基)甲烷抑制剂CBZ-Phe-Lys-CH2OCO-2，4，6-Me-3-Ph对牙周疼痛K的快速抑制作用是非常特异的。该抑制剂与与Arg特异的酶反应最快的多肽氯甲烷D-Phe-Pro-Arg-CHpCI和D-Phe-Phe-Arg-CH2Cl一起，可用于活性部位滴定纯化形式的酶和在完整的牙龈假单胞菌细胞、膜或部分中发现的酶的纯化形式。从这些滴定中可以明显看出，牙周疼痛R总是超过牙周疼痛K的大约3倍，并且牙周疼痛作为一个整体占了与细菌相关的蛋白分解活性的85%。通过化合物范围抑制动力学的阐明和牙周疼痛滴定方法的发展，将大大有助于今后对牙龈假单胞菌所阐述的蛋白酶的研究。
Porphyromonas gingivalis is one of the major pathogens associated with periodontal disease and releases powerful cysteine proteinases known as the gingipains, which are key virulence factors for this organism. The three forms of gingipains, gingipain R1, gingipain R2 (gingipain Rs) and gingipain K, which cleave specifically after arginine (R) or lysine (K) residues, were characterized in terms of the kinetics of their interaction with a wide range of synthetic peptidyl chloromethane inhibitors and a peptidyl (acyloxy)-methane. Chloromethane inhibitors were found to inhibit all the enzymes to varying degree dependent on the peptidyl components of the inhibitor. Thus, inhibitors containing a basic residue at P1 rapidly inactivated the gingipains and some specificity could be seen at the P2 site. The (acyloxy)methane inhibitor, Cbz-Phe-Lys-CH2OCO-2,4,6-Me-3-Ph, was very specific in its rapid inhibition of gingipain K over the gingipains R. This inhibitor, together with the peptidyl chloromethanes, D-Phe-Pro-Arg-CHpCI and D-Phe-Phe-Arg-CH2Cl, which reacted most rapidly with the Arg-specific proteinases, could be used to active site titrate purified forms of the enzymes and enzymes found in crude fractions such as intact P. gingivalis cells, vesicles or membrane fractions. From these titrations it was evident that gingipains R were always in an excess of about 3-fold over gingipain K and that the gingipains as a whole made up 85% of the proteolytic activity associated with the bacterium. The elucidation of the kinetics of inhibition by the range of compounds and the development of the titration method for gingipains will considerably aid in future studies on the proteases elaborated by P. gingivalis.