Abstract
Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC ). 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 (P(1)') 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 P(1)' 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 P(1)' 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 micrometer) and Glp-Asn-Pro-7-amido-4-methyl coumarin (K(i) = 0.97 micrometer).
Highlights
Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE)1 (EC 3.4.19.6) is a type II cell surface peptidase located on synaptosomal membranes in the central nervous system (1Ϫ4)
It can be seen from the representative HPLC traces shown in Fig. 1 that 1 mM thyrotropin-releasing hormone (TRH) was fully degraded after overnight incubation with TRH-DE
The results from this study show that alteration of the P1Ј residue in the tripeptide structure of TRH significantly affects both the ability of the resulting peptides to bind to TRH-DE, as measured by their Ki values, and the ability of TRH-DE to catalyze their hydrolysis
Summary
Pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHAMC) and 7-amino-4-methyl coumarin (AMC) were purchased from Bachem U. The rates of hydrolysis for each of those peptides identified as substrates were compared by measuring Glp production using microassays with shorter incubation times [39] In these assays, peptide (1 mM) was incubated at 37 °C in a total volume of 100 l, and incubation times and TRH-DE concentration were adjusted to produce a detectable amount of Glp while remaining within the initial rate period of the reaction. The initial rate of TRHAMC hydrolysis by TRH-DE was measured by incubating 5 M TRHAMC with 1.23 g of DPP-IV and 0.32 g of TRH-DE, both in the absence and presence of each library peptide (1 mM final concentration) under standard assay conditions [32]. The enzyme-peptide solution was subsequently added to a reaction mixture containing TRHAMC, DPP-IV, buffer, and peptide, and TRH-DE activity was measured using the continuous assay. All values are shown as the mean Ϯ S.D
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