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Heavy-atom isotope effects for enzymes of glutamine metabolism
Abstract
This study used isotope effects to examined the chemical mechanisms of the reactions catalyzed by Escherichia coli glutamine synthetase, asparaginase, and asparagine synthetase; enzymes which are important for synthesizing proteins and possessing therapeutic effects in the treatment of cancers. The glutaminase activity of asparaginase is 2-4% compared with normal asparaginase activity. The isotope effects on carbon-5 and the amide nitrogen of glutamine were determined ($\sp{13}$(V/K) and $\sp{15}$(V/K) are 1.0152 and 1.0189, respectively). The chemical step of the asparaginase reaction, the breaking of the carbon-nitrogen bond of glutamine, is the rate determining step. The glutaminase and synthetase activities catalyzed by asparagine synthetase were examined at pH 8 and 35$\sp\circ$C. In glutaminase experiments with only glutamine present, $\sp{13}$(V/K) and $\sp{15}$(V/K) are 1.0245 and 1.0095. In glutaminase experiments with ATP present, $\sp{13}$(V/K) and $\sp{15}$(V/K) are 1.0256 and 1.0065. In the synthetase experiment with aspartate and ATP present, $\sp{13}$(V/K) and $\sp{15}$(V/K) are 1.0231 and 1.0222. Carbon and nitrogen isotope effects on the glutaminase reaction catalyzed by asparagine synthetase and asparaginase are similar, indicating their chemical mechanisms are identical. Results for the asparagine synthetase reaction indicate a different mechanism. The carbon isotope effect is similar to those determined for deamidation by asparagine synthetase and asparaginase, which is most likely the result of the breaking of a carbon-nitrogen bond. $\sp{15}$(V/K) indicates that the amide nitrogen acts differently than in the glutaminase reactions. The nitrogen in the glutaminase reactions is being protonated at the same time as the carbon-nitrogen bond is broken. The large nitrogen isotope effect in the synthetase reaction indicates that nitrogen in the tetrahedral intermediate probably acts as a nucleophile on the carboxyl carbon of aspartyl-phosphate making asparagine. The carbon isotope effects determined for glutamine synthetase demonstrated that the chemical step is not the rate determining step.
Subject Area
Biochemistry
Recommended Citation
Stoker, Paul Wallace, "Heavy-atom isotope effects for enzymes of glutamine metabolism" (1994). ETD collection for University of Nebraska-Lincoln. AAI9507828.
https://digitalcommons.unl.edu/dissertations/AAI9507828