Titration and mapping of the active site of cysteine proteinases from Porphyromonas gingivalis (gingipains) using peptidyl chloromethanes.
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.
Potempa
J
J
Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Pike
R
R
Travis
J
J
D018829:Adhesins, Bacterial; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D010455:Peptides; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D018829:Adhesins, Bacterial; D015853:Cysteine Proteinase Inhibitors; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D010455:Peptides; D003546:Cysteine Endopeptidases