Potempa J, Pike R, Travis J
Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Biol Chem. 1997 Mar-Apr;378(3-4):223-30. doi: 10.1515/bchm.1997.378.3-4.223.
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-Me3-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-CH2Cl 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.
牙龈卟啉单胞菌是与牙周疾病相关的主要病原体之一，它能释放出强大的半胱氨酸蛋白酶，即牙龈蛋白酶，这些蛋白酶是该生物体的关键毒力因子。牙龈蛋白酶有三种形式，即牙龈蛋白酶R1、牙龈蛋白酶R2（牙龈蛋白酶Rs）和牙龈蛋白酶K，它们分别在精氨酸（R）或赖氨酸（K）残基后特异性切割，通过它们与多种合成肽基氯甲烷抑制剂和肽基（酰氧基）甲烷相互作用的动力学对其进行了表征。发现氯甲烷抑制剂能不同程度地抑制所有这些酶，这取决于抑制剂的肽基成分。因此，在P1位含有碱性残基的抑制剂能迅速使牙龈蛋白酶失活，并且在P2位可以看出一些特异性。（酰氧基）甲烷抑制剂Cbz-Phe-Lys-CH2OCO-2,4,6-Me3-Ph对牙龈蛋白酶K的快速抑制作用比对牙龈蛋白酶R更为特异。这种抑制剂与肽基氯甲烷D-Phe-Pro-Arg-CH2Cl和D-Phe-Phe-Arg-CH2Cl一起，它们与精氨酸特异性蛋白酶反应最快，可用于对纯化形式的酶以及在粗级分（如完整的牙龈卟啉单胞菌细胞、囊泡或膜级分）中发现的酶进行活性位点滴定。从这些滴定中可以明显看出，牙龈蛋白酶R的含量总是比牙龈蛋白酶K高出约3倍，并且牙龈蛋白酶总体上占与该细菌相关的蛋白水解活性的85%。对一系列化合物抑制动力学的阐明以及牙龈蛋白酶滴定方法的开发将极大地有助于未来对牙龈卟啉单胞菌产生的蛋白酶的研究。
