A Novel Assay Method to Determine the ?-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1.

ABSTRACT: Kynurenine aminotransferase 1 (KYAT1 or CCBL1) plays a major role in Se-methylselenocysteine (MSC) metabolism. It is a bi-functional enzyme that catalyzes transamination and beta-elimination activity with a single substrate. KYAT1 produces methylselenol (CH3SeH) via ?-elimination activities with MSC as a substrate. This methylated selenium compound is a major cytotoxic selenium metabolite, causing apoptosis in a wide variety of cancer cells. Methylselenol is volatile and possesses extraordinary nucleophilic properties. We herein describe a simple spectrophotometric assay by combining KYAT1 and thioredoxin reductase (TrxR) to detect CH3SeH in a coupled activity assay. The metabolite methylselenol and its oxidized form from MSC metabolism is utilized as a substrate for TrxR1 and this can be monitored spectroscopically at 340 nm. Our results show the feasibility of monitoring the ?-elimination of KYAT1 by our assay and the results were compared to the previously described ?-elimination assays measuring pyruvate. By using known inhibitors of KYAT1 and TrxR1, we further validated the respective reaction. Our data provide a simple but accurate method to determine the ?-elimination activity of KYAT1, which is of importance for mechanistic studies of a highly interesting selenium compound.

SUBMITTER: Selvam AK 

PROVIDER: S-EPMC7070735 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature