| Abstract
| - Oligopeptidase B is a member of a novel serine peptidase family, found in Gram-negativebacteria and trypanosomes. The enzyme is involved in host cell invasion, and thus, it is an importanttarget for drug design. Oligopeptidase B is specific for substrates with a pair of basic residues at positionsP1 and P2. The sensitivity of substrates to high ionic strength suggests that the arginines interact with thecarboxylate ions of the enzyme. On the basis of a three-dimensional model, two carboxyl dyads (Asp460and 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 substrateconcentrations (different inhibitory mechanisms were demonstrated with substrates bearing one and twoarginine residues), (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having onearginine, and (iv) interaction of Ca2+ with the dyads which simplified the complex pH dependence curves.Titration with a product-like inhibitor revealed the pKa 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 transitionstate of the reaction. Its role studied with the Tyr452Phe variant indicates that (i) the catalytic contributionof the OH group depends on the substrate and (ii) the catalysis is, unusually, an entropy-driven processat physiological temperature. The NH group of the scissile peptide bond accounts for the deviation of thereaction from the Eyring plot above 25 °C, and for abolishing potential nonproductive binding.
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