The role of amino acid side chains in decarboxylation reactions catalyzed by Pseudomonas cepacia 2,2-dialklglycine decarboxylates (EC 4.1.1.64), a pyridoxal 5'-phosphate-dependent enzyme which decarboxylates 2,2-dialklglycines and D-amino acids but transaminates L-amino acids, will be investigated using site directed mutagenesis. The function of active site histidine and lysine side chains will be studied by replacing these amino acids by alanine and glycine, respectively, and then using various small molecules to mimic the function of the lost side chains. These are prosthetic or auxiliary catalysts. This approach provides a direct test for the function of an active site side chain: If catalysis by the added molecules tends to be more efficient with molecules that are more basic, then the original side chain is likely to function as a base. If catalysis by the added molecules tends to be less efficient with molecules that are more basic, then the original side chain likely functions as an acid. A variety of small bases, first used by Toney and Kirsch to test the function of a lysine in aspartate aminotransferase ?Science 243, 1485 (1989)!, will be used to test the lysine of the dialklglycine decarboxylase. A series of imidazoles will be used to test the function of the active histidine in the same enzyme.
