Kinetic mechanism of Escherichia coli glutamine synthetase.
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The kinetic mechanism of Escherichia coli glutamine synthetase (unadenylylated) has been determined at pH 7.5, 25 °C. For both the forward and reverse biosynthetic reactions, initial velocity, product (forward reaction only), and dead-end inhibition patterns are consistent with preferred ordered addition of MgATP, glutamate, and ammonia, followed by the preferred ordered release of Pi, glutamine, and MgADP. These data and the previously published equilibrium isotope exchange data, when taken together, provide an overall mechanism that is steady-state random with strongly preferred ordered substrate addition and product release. Initial velocity data of 1 /v vs. 1/[NH4+] are nonlinear (concave downward) when glutamate is subsaturating but linear at saturating glutamate levels. These experiments suggest negatively cooperative binding of NH4+. This kinetic mechanism is in accord with the initial formation of the proposed 7-glutamyl phosphate intermediate from MgATP and glutamate, followed by ammonia attack to produce glutamine and Pi. Two new competitive inhibitors of E. coli glutamine synthetase, 3- amino-3-carboxypropanesulfonamide and 2-amino-4- phosphonobutyric acid, have been characterized. The low inhibition constants (Ki≃ 50 μM) of these glutamate analogues suggest that they may be structural mimics of a tetrahedral transition state in the catalytic mechanism. © 1980, American Chemical Society. All rights reserved.
author list (cited authors)
Meek, T. D., & Villafranca, J. J.