Development of a Continuous, Fluorometric Coupled Enzyme Assay for Thyrotropin-Releasing Hormone-Degrading Ectoenzyme
✍ Scribed by Julie A. Kelly; Gillian R. Slator; Keith F. Tipton; Carvell H. Williams; Karl Bauer
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 83 KB
- Volume
- 274
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4.19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80°C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave K m values for TRH-MCA of 3.4 ؎ 0.7 M (n ؍ 5) and 3.8 ؎ 0.5 M (n ؍ 5), respectively. K i values determined by the discontinuous assay for TRH and TRH-OH were 35 ؎ 4 M (n ؍ 3) and 311 ؎ 31 M (n ؍ 5), respectively.