Suneal K. Sreedharan

Catalytic site targeted mutagenesis of the α-gingivain gene of Porphyromonas gingivalis using Tn-4351 to generate isogenic mutants

The extracellular proteinases of the anaerobe Porphyromonas gingivalis, are implicated in the destruction of host defence mechanisms in periodontitis. We have previously purified one of these enzymes, alpha-gingivain, and established that it belongs to the cysteine proteinase family of enzymes. In the present study, transposon Tn4351 was used to alter the open reading frame encoding a region that includes the catalytic site of alpha-gingivain by targeted mutagenesis. Escherichia coli HB101 which harbours R751 was used to introduce the transposon into P. gingivalis ATCC 33277 by conjugal transfer. E. coli was transformed using the altered plasmid with a Cla I site insertion of a sequence common to the catalytic site histidine or cysteine of many cysteine proteinases. The frequency of the transconjugation was 4.5 x 10(5) while the recipient viable counts comprised 60% of the original P. gingivalis. The result of this targeted mutagenesis was inactivation of gingivains such that some colonies on skimmed-milk agar plates showed no clear surrounding zones of hydrolysis and their normal catalytic activity towards L-BAPNA was destroyed.

Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.

The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes.