Role of the oxyanion binding site and subsites S1 and S2 in the catalysis of oligopeptidase B, a novel target for antimicrobial chemotherapy.
Oligopeptidase B is a member of a novel serine peptidase family, found in Gram-negative bacteria and trypanosomes. The enzyme is involved in host cell invasion, and thus, it is an important target for drug design. Oligopeptidase B is specific for substrates with a pair of basic residues at positions P1 and P2. The sensitivity of substrates to high ionic strength suggests that the arginines interact with the carboxylate ions of the enzyme. On the basis of a three-dimensional model, two carboxyl dyads (Asp460 and Asp462 and Glu576 and Glu578) can be assigned as binding sites for arginines P1 and P2, respectively. The dyads are involved in several events: (i) substrate binding, (ii) substrate inhibition at high substrate concentrations (different inhibitory mechanisms were demonstrated with substrates bearing one and two arginine residues), (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having one arginine, and (iv) interaction of Ca2+ with the dyads which simplified the complex pH dependence curves. Titration with a product-like inhibitor revealed the pK(a) of the carboxyl group that perturbed the pH-kcat/Km profiles. The OH group of Tyr452 is part of the oxyanion binding site, which stabilizes the transition state of the reaction. Its role studied with the Tyr452Phe variant indicates that (i) the catalytic contribution of the OH group depends on the substrate and (ii) the catalysis is, unusually, an entropy-driven process at physiological temperature. The NH group of the scissile peptide bond accounts for the deviation of the reaction from the Eyring plot above 25 degrees C, and for abolishing potential nonproductive binding.
Juhász
Tünde
T
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 7, Budapest H-1518, Hungary.
Szeltner
Zoltán
Z
Renner
Veronika
V
Polgár
László
L
D000838:Anions / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D055672:Static Electricity
D000838:Anions; D000900:Anti-Bacterial Agents; D017931:DNA Primers; D012697:Serine Endopeptidases; C090703:oligopeptidase B; D010100:Oxygen