title: Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.
abstract: Although Arg-30, Asn-74, and Asn-79 appear totally conserved throughout the purF glutamine-dependent amidotransferases, their potential roles in catalysis and binding remain unexplored for any member of the enzyme family. Here we report the overexpression, purification, and kinetic characterization of a series of AS-B mutants which allow an examination of the functional roles of these 3 residues in glutamine-dependent nitrogen transfer. While Asn-79 appears to possess no catalytic function in AS-B, site-directed mutagenesis of Asn-74 has implicated this residue as playing a role in catalysis of nitrogen transfer from glutamine. The kinetic properties of the Asn-74 AS-B mutant enzymes appear consistent with both ammonia-mediated nitrogen transfer and two apparently novel mechanistic suggestions for this reaction involving either an oxyanion or imide intermediate (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). We also demonstrate that replacement of Arg-30 by an alanine residue yields an AS-B mutant for which the apparent Km for glutamine is increased in the glutamine-dependent synthesis of asparagine. In addition, ATP-dependent stimulation of the glutaminase activity of AS-B is modified or completely eliminated when Arg-30 is replaced by other amino acids. The latter observation may indicate the existence of a molecular switch involving Arg-30 which coordinates the two half-reactions catalyzed by the glutamine-dependent amidotransferases and synthetase domains of cellular AS-B.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
authors.forename: S K
authors.initials: SK
authors.lastname: Boehlein
authors.forename: N G
authors.initials: NG
authors.lastname: Richards
authors.forename: E S
authors.initials: ES
authors.lastname: Walworth
authors.forename: S M
authors.initials: SM
authors.lastname: Schuster
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001216:Asparagine / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001217:Aspartate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D009711:Nucleotides; D005973:Glutamine; D001216:Asparagine; D000641:Ammonia; D000255:Adenosine Triphosphate; D001120:Arginine; D000637:Transaminases; D001217:Aspartate-Ammonia Ligase; D009584:Nitrogen