Development of a satisfactory and general continuous assay for aminotransferases by coupling with (R)-2-hydroxyglutarate dehydrogenase

Abstract A continuous general spectrophotometric assay for measuring the activity of aminotransferases has been developed. It is based on the transamination of a keto compound (amino acceptor) and l -glutamate (amino donor), yielding the corresponding amino compound and 2-oxoglutarate. The rate of formation of 2-oxoglutarate is measured in a coupled reaction with overproduced recombinant nicotinamide adenine dinucleotide (NAD + )-dependent ( R )-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans , with the rate of absorbance decrease at 340 nm indirectly reflecting the aminotransferase activity. This new method allows continuous monitoring of the course of transamination. Because glutamate and 2-oxoglutarate are obligatory participants in most biological transamination reactions, a coupled assay based on measuring the formation of 2-oxoglutarate has very wide applicability. The article demonstrates its utility with branched-chain amino acid aminotransferase and l -valine:pyruvate aminotransferase.