SYNTHESIS OF ALPHA,BETA-DEUTERATED N-15 AMINO-ACIDS USING A COUPLED GLUTAMATE-DEHYDROGENASE BRANCHED-CHAIN AMINO-ACID AMINOTRANSFERASE SYSTEM

Gram amounts of various 15N-enriched L-amino acids have been synthesized using a coupled enzymatic system. Catalytic amounts of 15N-labeled L-glutamate are generated using (15NH4)2SO4, α-ketoglutarate, and glutamate dehydrogenase. The labeled glutamate in turn serves as an amine donor to an appropriate α-keto acid using the Escherichia coli branched-chain amino acid aminotransferase, the subcloning and overexpression of which is described. In order to drive the reaction to completion the redox cofactor NADPH is regenerated using a glucose dehydrogenase system. Since the aminotransferase catalyzes exchange of the α-hydrogen, carrying out this reaction in 2H2O gives rise to 2-2H,2-15N-labeled amino acids. Deuteration can be readily extended to the β position as well by preexchanging the α-keto acids in basic 2H2O. The isotopically labeled amino acids are recovered in yields of 70-80%. © 1993 Academic Press, Inc.