Production and properties of adhesin-free gingipain proteinase RgpA

Abstract

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.