Identification of the thyrotropin-releasing-hormone-degrading ectoenzyme as a metallopeptidase

A time-dependent inhibition of the thyrotropin-releasing-hormone (TRH)-degrading ectoenzyme (EC 3.4.19.-) from rat brain by the metal-complexing agents imidazole, NaCN, EDTA and 1,10-phenanthroline could be demonstrated. In contrast, the inhibition by the non-chelating analogues 4,7- and 1,7-phenanthroline was not time-dependent. At a concentration of 100 microM EDTA the enzymic activity decreased by 50% only after pretreatment for 6 h. It could be restored by addition of Zn2+, Ni2+ and Co2+ but not by other transition metal ions and also not by Ca2+ and Mg2+. Without pretreatment, the enzyme was activated by Co2+ and inhibited by Cu2+, Cd2+ and Hg2+ in a time-dependent manner, but remained unaffected by Ni2+ and Mn2+, as well as by Ca2+ and Mg2+. Compatible with the His-Glu-Xaa-Xaa-His consensus sequence of most zinc-containing metallopeptidases, chemical modification studies with carbodiimide revealed the presence of an essential acidic amino acid residue, probably located at the active site of the enzyme. The catalytically active metal ion could be exchanged for 65Zn and the enzyme could be effectively inhibited by L-pyroglutamyl hydroxamic acid, the chelating derivative of the TRH cleavage product pyroglutamic acid. The TRH-degrading ectoenzyme thus classifies as a member of the zinc-dependent metallopeptidase family.