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-enolpyruvylshikima te-3-phosphate (EPSP) synthase is reported. The H385N mutant is compar ed with wild type by a number of methods. H385N was found to retain 6% activity. Kinetic parameters, including K-m values for the natural su bstrates and K-i and K-d values for the inhibitor glyphosate, were fou nd to be similar to wild type. Unlike wild-type enzyme, H385N EPSP syn thase does not show accumulation of enzyme-bound product (EPSP) in the C-13 MMR spectrum under equilibrium conditions. These results suggest that this H385N mutant is less catalytically competent than the previ ously studied H385Q mutant and that the (NH)-H-epsilon of histidine ma y be involved in hydrogen bonding to another residue involved in compl exing the substrates/products. (C) 1996 Academic Press, Inc.