Characterization of proline endopeptidase from rat brain

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Abstract
A homogeneous proline endopeptidase from rat brain is characterized with respect to its substrate specificity and the residues essential for catalysis. The 2 fluorogenic substrate analogues tested.sbd.pyroglutamylhistidylprolyl-.beta.-naphthylamide and pyroglutamyl(N-benzylimidazolyl)-histidylprolyl-.beta.-naphthylamide.sbd.have higher Vmax values (19.5 and 26.9 .mu.mol .cntdot. min-1 .cntdot. mg-1, respectively), and considerably lower Km values (0.034 and 0.020 mM, respectively) than pyroglutamylhistidylprolylamide (Vmax = 2.9 .mu.mol.cntdot.min-1.cntdot.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, 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 (DFP) with an observed 2nd-order rate constant of 2.5 .times. 104 min-1 .cntdot. M-1. The stoichiometry of the alkylphosphorylation is 0.83 mol/mol of enzyme. The pH dependence of the inactivation by DFP implicates a residue(s) involved in covalent bond formation, having a pKa of 6.0. Proline endopeptidase is apparently a serine proteinase.

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