An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation.
The Fe(II)/2-oxoglutarate-dependent dioxygenases are a catalytically diverse family of nonheme iron enzymes that oxidize their primary substrates while decomposing the 2-oxoglutarate cosubstrate to form succinate and CO(2). We report a generic assay for these enzymes that uses succinyl-coenzyme A synthetase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide. We demonstrate the utility of this new method by measuring the kinetic parameters of two bacterial Fe(II)/2-oxoglutarate-dependent dioxygenases. Significantly, this method can be used to investigate both the productive turnover reactions and the nonproductive "uncoupled" decarboxylation reactions of this enzyme family, as demonstrated by using wild-type and variant forms of 2-oxoglutarate-dependent taurine dioxygenase. This assay is amenable to miniaturization and easily adapted to a format suitable for high-throughput screening; thus, it will be a valuable tool to study Fe(II)/2-oxoglutarate-dependent dioxygenases.
Luo
Lusong
L
Department of Enzymology and Mechanistic Pharmacology, MMPD CEDD, GlaxoSmithKline, Collegeville, PA 19426, USA.
Pappalardi
Melissa B
MB
Tummino
Peter J
PJ
Copeland
Robert A
RA
Fraser
Marie E
ME
Grzyska
Piotr K
PK
Hausinger
Robert P
RP
D049308:Dioxygenases / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry; D007501:Iron / Q000737:chemistry* / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011770:Pyruvate Kinase / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000378:metabolism; D019802:Succinic Acid / Q000032:analysis* / Q000378:metabolism
D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D009243:NAD; D019802:Succinic Acid; D007501:Iron; D007770:L-Lactate Dehydrogenase; D049308:Dioxygenases; D011770:Pyruvate Kinase; D013389:Succinate-CoA Ligases