EXPLORING THE ACTIVE-SITE OF CHORISMATE MUTASE BY COMBINATORIAL MUTAGENESIS AND SELECTION - THE IMPORTANCE OF ELECTROSTATIC CATALYSIS

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrange ment of chorismate to prephenate. Arg-90 in the active site of the enz yme from Bacillus subtilis is in close proximity to the substrate's et her oxygen and may contribute to efficient catalysis by stabilizing th e presumed dipolar transition state that would result upon scission of the C-O bond. To test this idea, we have developed a novel complement ation system for chorismate mutase activity in Escherichia coli by ree ngineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that fo r Cys-88, and active clones were selected. The results show that a pos itively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential, Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition s tate of the enzymatic chorismate rearrangement. The new selection syst em, in conjunction with combinatorial mutagenesis, renders the mechani sm of the natural enzyme(s) accessible to further exploration and open s avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.