Studies of substrate requirements, kinetic properties, and competive inhibitors of the enzymes catabolizing TRH in rat brain

Abstract

The enzymes catalyzing the breakdown of TRH are soluble and dependent upon active sulfhydryl groups. One of these enzymes is a pyroglutamyl aminopeptidase (EC 3.4.11.8) (apparent molecular weight 60,000 daltons) with a specificity restricted to pyroglutamyl peptide bonds. The second enzyme is a prolyl endopeptidase (apparent molecular weight 70,000 daltons) with a specificity restricted to proline peptide bonds wherein the proline is preceded by an alpha adjacent amino acid possessing a blocked N-terminus. Substrate requirements for this latter enzyme are identical to those reported previously for the TRH deamidase of rat brain, the prolyl endopeptidase of rabbit brain and the post proline cleaving enzyme of rat brain. We conclude that this enzymatic activity variously described as TRH deamidase, post proline cleaving enzyme, and prolyl endopeptidase is that of a single enzyme best denoted as a prolyl endopeptidase (EC 3.4.22.16, proposed). The pyroglutamyl aminopeptidase has a K m for TRH of 45 μM as compared to a K m for TRH of 2400 μM for the prolyl endopeptidase. Total enzymatic activity of the prolyl endopeptidase, however, is approximately 3500 nmol/h/rat brain with the total enzymatic activity of the pyroglutamyl aminopeptidase being approximately 600 nmol/h/rat brain. The 5- to 6-fold higher affinity of the pyroglutamyl aminopeptidase for TRH suggests that of these two catabolic pathways, removal of the N-terminal pyroglutamyl moiety is likely to be the more important pathway for the initial catabolism of TRH in rat brain. Analysis of substrate specificity permitted the synthesis of several effective competitive inhibitors of the two enzymes. Of these, the most effective inhibitors of the pyroglutamyl aminopeptidase wereπ-glu-NH 2 ( K i 185 μM) and π-glu-his- OCH 3 ( K i 16.5 μM). The most effective inhibitors of the prolyl endopeptidase wereAc-gly-pro-NH 2 ( K i 1143 μ M) and Z-gly-pro-NH 2 ( K i 442 μM).