Kinetic mechanism of beef pancreatic L-asparagine synthetase.
The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D009994:Osmolar Concentration; D010179:Pancreas / Q000201:enzymology*
D000643:Ammonium Chloride; D000255:Adenosine Triphosphate; D008025:Ligases; D001217:Aspartate-Ammonia Ligase