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J. Biol. Chem., Vol. 275, Issue 22, 16746-16751, June 2, 2000

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Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin-releasing Hormone-degrading Ectoenzyme for Thyrotropin-releasing Hormone-like Peptides^*

Julie A. Kelly^§, Gillian R. Slator, Keith F. Tipton, Carvell H. Williams^¶, and Karl Bauer

From the  Department of Biochemistry, Trinity College Dublin, Dublin 2, Ireland, the ^¶ School of Biology and Biochemistry, Medical Biology Centre, Queen's University, Belfast BT9 7BL, United Kingdom, and the  Max-Planck-Institut für experimentelle Endokrinologie, D-30603 Hannover, Germany

Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system. Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P₁') residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P₁' residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K_i values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P₁' residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, L-pyroglutamyl-L-asparaginyl-L-prolineamide (K_i = 17.5 µM) and Glp-Asn-Pro-7-amido-4-methyl coumarin (K_i = 0.97 µM).

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