Identification of catalytically important residues in the active site of Escherichia coli transaldolase

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A. E96A, T156A, and S176A were purified from a talB-defici ent host and analyzed with respect to their 3D structure and kinetic behavi or. X-ray analysis showed that side chain replacement did not induce unanti cipated structural changes in the mutant enzymes. Three mutations. N35A, E9 6A, and T156A resulted mainly in an effect on apparent k(cat), with little changes in apparent K-m values for the substrates. Residues N35 and T156 ar e involved in the positioning of a catalytic water molecule at the active s ite and the side chain of E96 participates in concert with this water molec ule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent K-m v alue for the donor substrate, fructose 6-phosphate, was increased nearly fi vefold while the apparent K-m value for the acceptor substrate, erythrose 4 -phosphate remained unchanged, consistent with a function for S176 in the b inding of the C1 hydroxyl group of the donor substrate. The mutant D17A sho wed a 300-fold decrease in k(cat), and a fivefold increase in the apparent K-m value for the acceptor substrate erythrose 4-phosphate, suggesting a ro le of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.