title: Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase.
abstract: Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.
authors.affiliation: Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
authors.forename: Rhona E
authors.initials: RE
authors.lastname: Feltzer
authors.forename: John O
authors.initials: JO
authors.lastname: Trent
authors.forename: Robert D
authors.initials: RD
authors.lastname: Gray
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D007477:Ions; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; C032923:microbial serine proteinases