The cysteine proteases of the periodontal pathogen Porphyromonas gingivalis known as the gingipains are considered the major virulence factors of this species. There are three gingipains two of which, RgpA and RgpB, are specific for cleavage of Arg-X peptide binds and one Kgp, specific for Lys-X peptide bonds. The gingipains have sequence similarity proposed to derive from their evolving following gene duplication events. RgpA and RgpB share 97% identity in the catalytic domain but are only 22% identical to the Kgp catalytic domain. Biochemical analysis and site directed mutation has indicated that Cys473 of RgpB is involved in catalysis. The crystal structure of RgpB determined in the presence of a small molecule inhibitor confirmed the involvement of this residue in catalysis and revealed His440 is a catalytic dyad partner. However, examination of the structure also indicated the acidic residue Glu381 may be also relevant for catalysis. In Kgp the comparable residue to Glu381 is the acidic residue Asp388. We have recently determined the structure of Kgp in the presence of a small molecule inhibitor. In this structure the Asp388 side-chain is positioned in a way that it could form a catalytic triad in conjunction with the identified catalytic Cys and His, reinforcing that the gingipains may function through a catalytic triad. In this study we produced a Glu381Ala mutation in RgpB. The mutation reduced RgpB activity to almost undetectable levels confirming Glu381 is essential for efficient catalysis by RgpB. The loss of RgpB function following the Glu381Ala mutation and gingipain structure information supports the contention that rather than a catalytic dyad the gingipains function via a catalytic Cys-His-Glu/Asp triad mechanism.