The H385N Mutant of 5-Enolpyruvylshikimate-3-phosphate Synthase: Kinetics, Fluorescence, and Nuclear Magnetic Resonance Studies

The site-directed mutagenesis of histidine-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The H385N mutant is compared with wild type by a number of methods. H385N was found to retain 6% activity. Kinetic parameters, includingKmvalues for the natural substrates andKiandKdvalues for the inhibitor glyphosate, were found to be similar to wild type. Unlike wild-type enzyme, H385N EPSP synthase does not show accumulation of enzyme-bound product (EPSP) in the13C NMR spectrum under equilibrium conditions. These results suggest that this H385N mutant is less catalytically competent than the previously studied H385Q mutant and that the NϵH of histidine may be involved in hydrogen bonding to another residue involved in complexing the substrates/products.