Identification of arginine residues in the putative L-aspartate binding site of Escherichia coli adenylosuccinate synthetase.
Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis. The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics. CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable. However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme. Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate. The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305.
Wang
W
W
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Poland
B W
BW
Honzatko
R B
RB
Fromm
H J
HJ
D000265:Adenylosuccinate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D014157:Transcription Factors
D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D001224:Aspartic Acid; D001120:Arginine; D000265:Adenylosuccinate Synthase