5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE OF BACILLUS-SUBTILIS IS AN ALLOSTERIC ENZYME - ANALYSIS OF ARG24-]ASP, PRO105-]SER AND HIS385-]LYS MUTATIONS SUGGESTS A HIDDEN PHOSPHOENOLPYRUVATE-BINDING SITE

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has b een cloned, expressed and purified to near homogeneity. Clustal alignm ent of the amino acid sequences from different bacteria revealed sever al conserved residues located in the N-terminal, middle and C-terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis. Steady-state kinetic anal ysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimat e-3-phosphate synthase enzymes investigated so far. Both substrates, p hosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multipl e interaction sites. There are two sites for P-pyruvate binding, catal ytic and non-catalytic. Glyphosate (N-phosphonomethyl glycine) compete s for binding at the catalytic site and does not interact at the secon dary site. Glyphosate in the absence of ammonium ions increases cooper ativity of P-pyruvate binding and favors dimerization of the enzyme th rough an interaction between P-pyruvate-binding sites. The ammonium-io n-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no co operativity with respect to P-pyruvate. Absence of ammonium ions decre ases affinity for substrates and introduces cooperativity. Cooperativi ty was also introduced in the enzyme by point mutations, Arg24-->Asp a nd His385-->Lys. The latter mutant of the native enzyme exists as a di mer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstra ted by staining for the enzyme activity on the native gel and by resol ving purified enzyme preparations on a sucrose density gradient. A mod el describing the alteration in the aggregation status of the enzyme b y the inhibitor, activator and the substrates has been proposed.