Characterization of proline endopeptidase from rat brain.
A homogeneous proline endopeptidase from rat brain is characterized with respect to its substrate specificity and the residues essential for catalysis. The two fluorogenic substrate analogues tested, pyroglutamylhistidylprolyl-beta-naphthylamide and pyroglutamy(N-benzylimidazolyl)-histidylprolyl-beta-naphthylamide, have higher Vmax values (19.5 and 26.9 mumol . min-1 . mg-1, respectively) and considerably lower Km values (0.034 and 0.020 mM, respectively) than pyroglutamylhistidylprolylamide (Vmax = 2.9 mumol . min-1 . mg-1 and Km = 4.1 mM). Both fluorogenic substrates give rise to pH optima and pH-rate profiles similar to those of the amide. Values of Km and kcat are determined as a function of pH. Km is pH independent, with the titration curve for kcatKm-1 implicating an active-site residue(s) with a pKa of 6.2. Proline endopeptidase can be completely inactivated by low concentrations of diisopropyl fluorophosphate with an observed second-order rate constant of 2.5 x 10(4) min-1 . M-1. The stoichiometry of the alkylphosphorylation is 0.83 mol/mol of enzyme. The pH dependence of the inactivation by diisopropylfluorophosphate implicates a residue(s) involved in covalent bond formation having a pKa of 6.0. These data suggest that proline endopeptidase is a serine proteinase.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D000085822:Prolyl Oligopeptidases; D011485:Protein Binding; D051381:Rats; D012697:Serine Endopeptidases; D013116:Spinal Cord / Q000201:enzymology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives; D014018:Tissue Distribution
D013439:Sulfhydryl Reagents; D007531:Isoflurophate; D013973:Thyrotropin-Releasing Hormone; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D007460:Iodoacetamide