Studies of the ammonia-dependent reaction of beef pancreatic asparagine synthetase

Abstract

We have studied the asparagine synthetase reaction with regard to the ammonia-dependent production of asparagine. Hydroxylamine was shown to be an alternate substrate for the asparagine synthetase reaction, and some of its kinetic properties were examined. The ammonia-dependent reaction was examined with regard to inhibition by asparagine. It was found that asparagine inhibition was partial competitive with respect to ammonia, regardless of the concentration of aspartate. However, when MgATP was not saturating, the inhibition by asparagine became linear competitive. These results were interpreted to be consistent with a kinetic mechanism for asparagine synthetase where ammonia is bound to the enzyme followed by MgATP causing asparagine release.

Asparagine Synthetase Chemotherapy

“…The detailed kinetic and structural characterization of ASNS from mammalian sources historically proved to be difficult because of both reported low abundance and instability of the native enzyme during purification (25)(26)(27)(28)(29). In addition, only small amounts of recombinant, wild-type human ASNS could be obtained in a variety of early expression systems, the enzyme being purified to homogeneity using an affinity chromatography protocol (30)(31)(32).…”

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

“…The second group of asparagine synthetases, on the other hand, is present in both prokaryotes and eukaryotes and employs glutamine as the predominant source of nitrogen in obtaining asparagine from aspartate and ATP (Reaction 2), although ammonia can be employed as an alternative to glutamine (7)(8)(9). In addition, this class of synthetases acts as glutaminases in the absence of aspartate (Reaction 3).…”

Crystallization and preliminary crystallographic study of asparagine synthetase fromEscherichia coli

“…6.3.5.4] is found not only in prokaryotes (Humbert & Simoni, 1980;Reitzer & Magasanik, 1982) but also in eukaryotes (Andrulis, Chen & Ray, 1987;Gantt & Arfin, 1981;Hongo & Sato, 1981 ;, and encoded by the asnB gene. Studies of steady-state kinetic mechanisms for both enzymes have demonstrated that the mechanism of the ammonia-dependent enzymes is different from that of the glutamine-dependent enzymes (Cedar & Schwartz, 1969b;Hongo & Sato, 1985;Mehlhaff, Luehr & Schuster, 1985). Both types of asparagine synthetase genes have been cloned and sequenced from E. coli (Nakamura et al, 1981;Scofield, Lewis & Schuster, 1990), and asnB-type from the human enzyme as well (Andrulis, Chen & Ray, 1987).…”