Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli

Structural prediction of several bacterial and plant ADP-glucose pyrophosph orylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized p yrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., C ambillau, C., and Bourne, Y. (1999) EMBO J. 18,4096-4107; Blankenfeldt, W., Asuncion, M., Lam, J. S., and Naismith, J. H. (2000) EMBO J. 19, 6652-6663 ). This comparison led to the discovery of highly conserved residues throug hout the superfamily of pyrophosphorylases despite the low overall homology . One of those residues, Asp(142) in the ADP-glucose pyrophosphorylase from Escherichia coli, was predicted to be near the substrate site. To elucidat e the function that Asp(142) might play in the E. coli ADP-glucose pyrophos phorylase, aspartate was replaced by alanine, asparagine, or glutamate usin g site-directed mutagenesis. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specifi c activity of up to 4 orders of magnitude. Comparison of other kinetic para meters, i.e. the apparent affinities for substrates and allosteric effector s, showed no significant changes, excluding this residue from the specific role of ligand binding. Only the D142E mutant exhibited altered K-m values but none as pronounced as the decrease in specific activity. These results show that residue Asp(142) is important in the catalysis of the ADP-glucose pyrophosphorylase from E. coli.