Glutamine inhibits the ammonia-dependent activities of two Cys-1 mutants of human asparagine synthetase through the formation of an abortive complex.
Cys-1 mutants of recombinant human asparagine synthetase were constructed and their ability to catalyze the glutamine-dependent nitrogen transfer reaction required for asparagine biosynthesis was determined. In agreement with previous work, altering Cys-1 to either Ala or Ser eliminated the glutamine-dependent activity while only minimally affecting the kinetic properties of the ammonia-dependent reaction. A lack of glutaminase activity in these mutants also allowed examination of glutamine binding in studies of the ability of glutamine to inhibit the ammonia-dependent production of asparagine. In both mutants, analysis of the observed kinetics indicated that glutamine inhibited ammonia-dependent asparagine synthesis through the formation of an abortive complex. This unanticipated observation suggests that the commonly accepted mechanism for nitrogen transfer from the primary amide of glutamine to aspartic acid in asparagine synthetase may have to be re-examined. A novel mechanistic proposal which is consistent with the formation of an abortive complex in the two Cys-1 mutants is presented.
Sheng
S
S
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610.
Moraga-Amador
D A
DA
van Heeke
G
G
Allison
R D
RD
Richards
N G
NG
Schuster
S M
SM
D000641:Ammonia / Q000378:metabolism*; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D005973:Glutamine / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D005973:Glutamine; D000641:Ammonia; D004247:DNA; D001217:Aspartate-Ammonia Ligase; D003545:Cysteine