Production and properties of adhesin-free gingipain proteinase RgpA

The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase–adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpA(cat) as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-l-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of K(m), V(max), k(cat) and k(cat)/K(m) for each enzyme were similar, but with glycylglycine K(m) decreased, V(max) increased and k(cat) increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The k(cat)/K(m) for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with K(i) 13 nM and 15 nM K(i) respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with K(i) 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA.