Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli.
Structural prediction of several bacterial and plant ADP-glucose pyrophosphorylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized pyrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, 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 throughout 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 elucidate the function that Asp(142) might play in the E. coli ADP-glucose pyrophosphorylase, aspartate was replaced by alanine, asparagine, or glutamate using site-directed mutagenesis. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specific activity of up to 4 orders of magnitude. Comparison of other kinetic parameters, i.e. the apparent affinities for substrates and allosteric effectors, 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.
Frueauf
J B
JB
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Ballicora
M A
MA
Preiss
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D001224:Aspartic Acid; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase