Conductometric method for the rapid characterization of the substrate specificity of amine-transaminases.
Amine-transaminases (ATAs, omega-transaminases, omega-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and well-known amino acid-transaminases, ATAs are able to convert substrates lacking an alpha-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate alpha-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrically since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 microS mM(-1). In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis.
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Strasse 4, 17487 Greifswald, Germany.
Sebastian
S
Schätzle
Matthias
M
Höhne
Karen
K
Robins
Uwe T
UT
Bornscheuer
D000588:Amines / Q000378:metabolism*; D002138:Calibration; D002384:Catalysis; D002849:Chromatography, Gas; D004553:Electric Conductivity; D007700:Kinetics; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
D000588:Amines; D000637:Transaminases