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PURIFICATION AND MECHANISTIC STUDIES OF BEEF PANCREATIC ASPARAGINE SYNTHETASE
Abstract
A method for measuring asparagine synthetase activity was developed, based on the decarboxylation of the (beta)-carboxyl group of L-{4-('14)C} aspartate in the presence of pyridoxal and Al('+3) ions. The (beta)-amido group of L-{4-('14)C} asparagine remains intact under the same conditions. Bovine pancreatic L-asparagine synthetase was purified 1,400-fold to a specific activity of 170nmoles Asn/min./mg. Ammonium sulfate fractionation, DEAE ion exchange and Cibacron blue affinity chromatographies, and new methods of HPLC ion exchange and size exclusion chromatography were utilized. The enzyme was found to have a molecular weight of 110,000 - 120,000 by HPLC size exclusion. Asparagine synthetase catalyzes the formation of asparagine from aspartate with the hydrolysis of ATP to AMP and PP(,i), glutamine or ammonia being the nitrogen donor. For the ammonia-dependent reaction, it was found that ammonia binds first, followed by a random addition of aspartate and ATP. The products were released in an ordered manner: pyrophosphate, AMP, and asparagine. Asparagine was found to be a partial competitive inhibitor with respect to both ammonia and the alternate substrate, hydroxylamine. This could be due to a regulatory binding site for asparagine or to asparagine interfering by binding in the active site but still allowing ammonia to bind. Chloride was found to inhibit the ammonia-dependent reaction competitively and produce negative cooperativity with respect to ammonia. The glutaminase activity of the enzyme was co-purified through the purification steps and was inhibited by aspargine, aspartate, ATP, and hydroxylamine. It was found that ammonia was released first followed by glutamate. This provides support for the thought that the synthetase and glutaminase activities occur at the same site on the enzyme as opposed to the current thought that they are at different sites with the ammonia being shuttled during the synthetase reaction. An aspartyl-AMP intermediate was identified for the mammalian asparagine synthetase reaction. The ('18)O isotope was found to be transferred from aspartate to the AMP phosphorus but not to pyrophosphate as evidenced by the ('18)O shift of ('31)P-NMR spectra.
Subject Area
Biochemistry
Recommended Citation
LUEHR, CRAIG ALAN, "PURIFICATION AND MECHANISTIC STUDIES OF BEEF PANCREATIC ASPARAGINE SYNTHETASE" (1983). ETD collection for University of Nebraska-Lincoln. AAI8308788.
https://digitalcommons.unl.edu/dissertations/AAI8308788