Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K4 11R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kine tic characterization of mutants R100K, K340R, and E418A, which retain activ ity, is reported. Of those, R100K and K430R do not accumulate enzyme interm ediate of enzyme-bound product under equilibrium conditions. These residues , while not essential for catalysis, are most likely important for substrat e binding. All of the mutants are shown to be correctly folded by NMR spect roscopy.