Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas.
Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.
D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000378:metabolism*; D005973:Glutamine / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008433:Mathematics; D051379:Mice; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity
D005973:Glutamine; D008025:Ligases; D001217:Aspartate-Ammonia Ligase