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Pure Appl. Chem., 2000, Vol. 72, No. 3, pp. 373-384

Use of inhibitors to study reactions catalyzed by enzymes requiring pyridoxal phosphate as coenzyme

Benjamin Adams, B. Svante Axelsson, Kenneth J. M. Beresford, Nicola J. Church, Philip A. Spencer, Sheena M. Whyte and Douglas W. Young*

Sussex Centre for Biomolecular Design and Drug Development, University of Sussex, Falmer, Brighton, BN1 9QJ, UK

Abstract: The stereochemistry of a variety of pyridoxal phosphate-mediated enzymic reactions has been studied using enzyme inhibitors that are stereospecifically labeled in the β-position with deuterium. A versatile synthesis has been developed to prepare a wide variety of stereospecifically labeled d- and l-amino acids and inhibitors. Investigation of the "turnover" of β-chloro-d-alanine and d- and l-serine-O-sulfate by d-amino acid aminotransferase and l-aspartate aminotransferase respectively has shown that reaction within the active site of the former enzyme occurs with retention of stereochemistry. Although l-aspartate aminotransferase is an enzyme of the α-family, when it was incubated with β-chloro-l-alanine in the presence of 2-mercaptoethanol, β-substitution occurred. This was shown to involve retention of stereochemistry, an outcome typical of reactions catalyzed by enzymes of the β-family that have little or no homology with enzymes of the α-family. Formation of the "Schnackerz intermediate" has been studied as has the d-amino acid oxidase catalyzed reaction of the naturally occurring inhibitor d-propargylglycine.