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

Chemical modification of maize starch synthase IIb-2 (SSIIb-2) using 1-ethy l-3-(3-dimethy-laminopropyl)carbodiimide (EDAC), which modifies acidic amin o acid residues, resulted in a time- and concentration-dependent inactivati on of SSIIb-2. ADPG1c was found to completely protect SSIIb-2 from inactiva tion by EDAC. These results suggest that glutamate or aspartate is importan t for SS activity. On the basis of the sequence identity of SS, conserved a cidic amino acids were mutagenized to identify the specific amino acid resi dues important for SS activity. Three amino acids (D21, D139, and E391) wer e found to be important for SS activity. D21N showed 4% of the wild-type en zyme 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 ch ange in affinity for ADPG1c, suggesting that the negative charge is importa nt for ADPG1c binding. When sites D139 and E391 were changed to their respe ctive 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 subst rates. 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 am ylopectin. The circular dichroism analysis indicates that these kinetic cha nges may not be due to a major conformation change in the protein. These re sults provide the first evidence that the conserved aspartate and glutamate residues could be involved in the catalysis or substrate binding of SS.