NMR determination of hydrogen transfer stereospecificity of alpha-keto acid dehydrogenase complexes: a one-step...

A simple one-step method is described for the determination of hydrogen transfer stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreductases. Alcohol and lipoamide dehydrogenases whose stereospecificities are known are employed to prepare stereospecifically deuterated reduced nicotinamide adenine dinucleotide which is immediately reoxidized in situ in a NMR tube by the enzyme under investigation. Alternatively, the reduced nicotinamide adenine dinucleotide produced by the test enzyme is reoxidized in situ by enzymes of known stereospecificity, such as glutamate and lactate dehydrogenases. The reoxidized coenzyme of the coupled reactions is analyzed for its deuterium content by NMR. The presence and absence of the absorption band due to the hydrogen or deuterium, respectively, at the 4-position of the nicotinamide ring is used to diagnose the stereospecificity of the test enzyme. Application of this direct in situ coupling method to dihydrolipoyl dehydrogenases of Escherichia coli pyruvate and alpha-ketoglutarate dehydrogenase complexes indicates that the enzymes are B-side stereospecific for nicotinamide adenine dinucleotide.