Involvement of conserved aspartate and glutamate residues in the catalysis and substrate binding of maize starch synthase

Chemical modification of maize starch synthase IIb-2 (SSIIb-2) using 1-ethyl-3-(3-dimethy-laminopropyl)carbodiimide (EDAC), which modifies acidic amino acid residues, resulted in a time- and concentration-dependent inactivation of SSIIb-2. ADPG1c was found to completely protect SSIIb-2 from inactivation by EDAC. These results suggest that glutamate or aspartate is important for SS activity. On the basis of the sequence identity of SS, conserved acidic amino acids were mutagenized to identify the specific amino acid residues important for SS activity. Three amino acids (D21, D139, and E391) were found to be important for SS activity. D21N showed 4% of the wild-type enzyme activity and a 10-fold decrease in the affinity for ADPG1c, while the conservative change from D21 to E resulted in a decrease in V-max and no change in affinity for ADPG1c, suggesting that the negative charge is important for ADPG1c binding. When sites D139 and E391 were changed to their respective amide form, no SS activity was detected. With the conservative change, D139E showed a decrease in V-max and no changes in apparent K-m for substrates. E391D showed a 9-fold increase in K-m for ADPG1c, a 12-fold increase in apparent K-m for glycogen, and a 4-fold increase in apparent K-m for amylopectin. The circular dichroism analysis indicates that these kinetic changes may not be due to a major conformation change in the protein. These results provide the first evidence that the conserved aspartate and glutamate residues could be involved in the catalysis or substrate binding of SS.