Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase.
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.
Shuttleworth
W A
WA
Department of Biochemistry and Biophysics, Washington State University, Pullman, 99164-4660.
Evans
J N
JN
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007554:Isotopes; D006639:Histidine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase