The topology of the glutamine and ATP binding sites of human asparagine synthetase.
Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.
Department of Chemistry, University of Nebraska-Lincoln 68588-0304.
M C
MC
Larsen
S M
SM
Schuster
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D001386:Azides / Q000494:pharmacology; D003001:Cloning, Molecular; D003980:Diazooxonorleucine / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012441:Saccharomyces cerevisiae; D014466:Ultraviolet Rays
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001386:Azides; D011994:Recombinant Proteins; D003980:Diazooxonorleucine; D005973:Glutamine; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D001217:Aspartate-Ammonia Ligase