SITE-DIRECTED MUTAGENESIS AND NMR-STUDIES OF HISTIDINE-385 MUTANTS OF5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE

The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-p hosphate (EPSP) synthase is reported. The steady-state kinetics for tw o mutants, H385Q and H385A, are compared with that of the wild-type en zyme. H385Q EPSP synthase was found to have 25% wild-type enzyme activ ity, whereas H385A EPSP synthase retained 1% activity. The K-M values for P-i and shikimate 3-phosphate were unaffected, whereas the K-M for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synth ase. The K-M for EPSP was unaffected in H385Q but raised by a factor o f 10 in H385A EPSP synthase. The binding of glyphosate was studied by fluorescence spectroscopy and by P-31 NMR Spectroscopy Direct observat ion of the enzyme-intermediate complexes by C-13 NMR spectroscopy with [2,3-C-13]phosphoenolpyruvate was studied for the mutant enzymes and compared with the wild type. Under equilibrium conditions, H385A EPSP synthase does not accumulate enzyme-bound EPSP. These results suggest that, while critically located in the PEP binding site, His-385 is not the residue responsible for initiating catalysis through the protonat ion of PEP.