title: Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase.
abstract: Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.
authors.affiliation: Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
authors.forename: F
authors.initials: F
authors.lastname: Liu
authors.forename: Q
authors.initials: Q
authors.lastname: Dong
authors.forename: H J
authors.initials: HJ
authors.lastname: Fromm
mesh_terms: D000265:Adenylosuccinate Synthase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*
chemical_list: D010710:Phosphates; D004247:DNA; D000265:Adenylosuccinate Synthase