title: 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 340nm 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.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Xuejing
authors.initials: X
authors.lastname: Yu
authors.forename: Julia
authors.initials: J
authors.lastname: Bresser
authors.forename: Iris
authors.initials: I
authors.lastname: Schall
authors.forename: Ivana
authors.initials: I
authors.lastname: Djurdjevic
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Xingguo
authors.initials: X
authors.lastname: Wang
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D045850:Acidaminococcus / Q000201:enzymology; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D007656:Ketoglutaric Acids; D009243:NAD; D018698:Glutamic Acid; D000429:Alcohol Oxidoreductases; C023858:2-hydroxyglutarate dehydrogenase; D000637:Transaminases