Alkaline proteinase inhibitor of Pseudomonas aeruginosa. Interaction of native and N-terminally truncated inhibitor proteins with Pseudomonas metalloproteinases.

The apr locus of Pseudomonas aeruginosa encodes alkaline proteinase (APR), a member of the metzincin metalloendopeptidase superfamily, and an 11.4-kDa alkaline proteinase inhibitor (APRin). We describe here the expression in Escherichia coli and characterization of full-length and N-terminally truncated APRin proteins. Fluorescence and circular dichroism spectra indicated that the recombinant proteins were folded into native-like structures. Analytical ultracentrifugation showed that APRin was monomeric and formed a 1:1 complex with APR. Binding of wild-type APRin to APR occurred with association (k(on)) and dissociation (k(off)) rate constants of 0.29 +/- 0.06 x 10(6) m(-1) s(-1) and 1.15 +/- 0.08 x 10(-6) s(-1) to give an equilibrium dissociation constant (K(D)) of approximately 4 x 10(-12) m (25 degrees C, pH 7.0, ionic strength 2.4 m). The association rate decreased by approximately 2-fold in 20% glycerol and increased by approximately 3-fold in 0.1 m NaCl. The glycerol effect suggests a diffusion-limited reaction, and the small salt effect indicates that electrostatic interactions contribute little to binding. Deletion of residues 1-10, 1-6, or 6-10 abolished inhibition, and deletion of residues 1-2, 1-3, 1-4, and 1-5 resulted in a progressively decreased affinity of APRin for APR (K(D) = 0.12 micrometer the Delta(1-5) mutant). Substitution of APRin residues 6-10 with a (Gly)(5) or (Pro)(5) linker restored inhibitory activity of the Delta(6-10) mutant but with a 100- and 50-fold reduction in K(D). Log k(on) for the full-length and truncated inhibitors correlated with the solvent-accessible surface area of their N-terminal regions, suggesting that increased interactions and/or desolvation of these residues in the transition state for binding contribute to the enhanced association rate. Treatment of APRin with pseudolysin, also secreted by P. aeruginosa, resulted in removal of residues 1-5. APRin was neither an inhibitor nor a substrate of other metzincins, including collagenase or gelatinases A or B.