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 α chain of ∼54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked α and β 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 Nα-benzoyl-l-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 °C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 °C of 6.5±0.05 and 9.7±0.05, and ΔH values of ∼29 kJ/mol and ∼ 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 α/β 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.

This content is only available as a PDF.

Author notes

Present address: Department of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

Present address: PerSeptive Biosystems UK Ltd., 3 Harforde Court, Foxholes Business Park, Hertford, SG13 7NW, U.K.