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-dimethylaminopropyl)carbodiimide (EDAC), which modifies acidic amino acid residues, resulted in a time- and concentration-dependent inactivation of SSIIb-2. ADPGlc 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 ADPGlc, while the conservative change from D21 to E resulted in a decrease in V(max) and no change in affinity for ADPGlc, suggesting that the negative charge is important for ADPGlc 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 ADPGlc, 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.
Nichols
D J
DJ
Interdepartmental Plant Physiology Major and ExSeed Genetics L.L.C., 1567 Food Science Building, Iowa State University, Ames, Iowa 50011-1061, USA.
Keeling
P L
PL
Spalding
M
M
Guan
H
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002234:Carbodiimides / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D017386:Sequence Homology, Amino Acid; D013214:Starch Synthase; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D002234:Carbodiimides; D010940:Plant Proteins; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D001224:Aspartic Acid; D018698:Glutamic Acid; D005964:Glucosyltransferases; C111377:starch synthase II; D013214:Starch Synthase