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

The stereochemistry of a variety of pyridoxal phosphate-mediated enzymic re actions has been studied using enzyme inhibitors that are stereospecificall y labeled in the beta-position with deuterium. A versatile synthesis has be en developed to prepare a wide variety of stereospecifically labeled D- and L-amino acids and inhibitors. Investigation of the "turnover" of beta-chlo ro-D-alanine and D- and L-serine-O-sulfate by D-amino acid aminotransferase and L-aspartate aminotransferase respectively has shown that reaction with in the active site of the former enzyme occurs with retention of stereochem istry. Although L-aspartate aminotransferase is an enzyme of the alpha-fami ly, when it was incubated with beta-chloro-L-alanine in the presence of 2-m ercaptoethanol, beta-substitution occurred. This was shown to involve reten tion of stereochemistry, an outcome typical of reactions catalyzed by enzym es of the beta-family that have little or no homology with enzymes of the a lpha-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.