EVIDENCE FOR THE SHIKIMATE-3-PHOSPHATE INTERACTING SITE IN THE N-TERMINAL DOMAIN OF 5-ENOLPYRUVYL SHIKIMATE-3-PHOSPHATE SYNTHASE OF BACILLUS-SUBTILIS

The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EP SPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substi tuted by glutamic acid and aspartic acid residues respectively by site -directed mutagenesis. Native and the mutant proteins were expressed u sing a two-vector system and the expressed proteins were purified to n ear homogeniety. The replacement of either Lys 19 or Arg 24 with a neg atively charged residue nearly completely abolished the enzyme activit y. The kinetic characterization of the purified wild type and the muta nt proteins revealed that the substitution of positively charged resid ues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.