Shuttleworth W A, Evans J N
Department of Biochemistry and Biophysics, Washington State University, Pullman, 99164-4660.
Biochemistry. 1994 Jun 14;33(23):7062-8. doi: 10.1021/bi00189a007.
The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The steady-state kinetics for two mutants, H385Q and H385A, are compared with that of the wild-type enzyme. H385Q EPSP synthase was found to have 25% wild-type enzyme activity, whereas H385A EPSP synthase retained 1% activity. The KM values for Pi and shikimate 3-phosphate were unaffected, whereas the KM for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synthase. The KM for EPSP was unaffected in H385Q but raised by a factor of 10 in H385A EPSP synthase. The binding of glyphosate was studied by fluorescence spectroscopy and by 31P NMR spectroscopy. Direct observation of the enzyme-intermediate complexes by 13C NMR spectroscopy with [2,3-13C]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 protonation of PEP.
