5-Enolpyruvylshikimate-3-phosphate Synthase of Bacillus subtilis is an Allosteric Enzyme. Analysis of Arg24Asp, Pro105Ser and His385Lys mutations suggests a hidden phosphoenol pyruvate-binding site

Abstract

5-Enol pyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several 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 analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enol pynivylshikimate-3-phosphate synthase enzymes investigated so far. Both substrates, phosphoenol pyruvate (P -pyruvate) and shikimate 3-phosphate have multiple interaction sites. There are two sites for P -pyruvate binding, catalytic and non-catalytic. Glyphosate (N -phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P -pyruvate binding and favors dimerization of the enzyme through an interaction between P -pyruvate-binding sites. The ammonium-ion-activated 5-enol pyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24Asp and His385Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.