title: Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor.
abstract: Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.
authors.affiliation: MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 32 Newark Street, London E1 2AA, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Rangarajan
authors.forename: S J
authors.initials: SJ
authors.lastname: Smith
authors.forename: S
authors.initials: S
authors.lastname: U
authors.forename: M A
authors.initials: MA
authors.lastname: Curtis
mesh_terms: D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D019143:Evolution, Molecular; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014774:Virulence
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D007461:Iodoacetates; D011480:Protease Inhibitors; D001120:Arginine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D007930:Leucine; C024974:E 64; D019807:Iodoacetic Acid; D007460:Iodoacetamide