KINETIC MECHANISM OF ESCHERICHIA-COLI ASPARAGINE SYNTHETASE-B

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesi s of asparagine from aspartate, glutamine, and ATP. A combination of k inetic, isotopic-labeling, and stoichiometry studies have been perform ed to define the nature of nitrogen transfer mediated by AS-B. The res ults of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The in itial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibi tion studies were only consistent with the latter pathway. Moreover, i sotope-trapping studies confirmed that the enzyme-ATP-aspartate comple x was kinetically competent. Studies using O-18-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and st oichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl- AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.