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

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.