Kinetic mechanism of beef pancreatic L-asparagine synthetase

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Abstract
The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of 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. PPi, 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. PPi, AMP and asparagine were then sequentially released as in the glutamine-utilizing mechanism. A comprehensive mechanism has been proposed through which glutamine or NH3 can provide nitrogen for asparagine production from aspartate.