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 elec...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-06, Vol.270 (22), p.13160-13163
Main Authors: Wang, W, Poland, B W, Honzatko, R B, Fromm, H J
Format: Article
Language:English
Subjects:
Adenylosuccinate Synthase - genetics
Adenylosuccinate Synthase - isolation & purification
Adenylosuccinate Synthase - metabolism
Amino Acid Sequence
Arginine - metabolism
Aspartic Acid - metabolism
Base Sequence
Binding Sites
Cyclic AMP Response Element-Binding Protein
DNA, Complementary
DNA-Binding Proteins - genetics
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Secondary
Transcription Factors
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Summary: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.
ISSN:0021-9258
DOI:10.1074/jbc.270.22.13160