Escherichia coli glutamine synthetase. Determination of rate-limiting steps by rapid-quench and isotope partitioning experiments. Academic Article uri icon


  • The ATPase and biosynthetic reactions of unadenylylated Escherichia coli glutamine synthetase have been studied by using rapid-quench, stopped-flow, and isotope partitioning techniques. The time course of the ATPase reaction (24 °C) is characterized by a “burst” of acid-labile phosphate equivalent to 0.67 mol of enzyme, which is followed by a slower steady-state phase. In the presence of Mg2+, only unadenylylated subunits are capable of producing the observed burst. The rate constant for the transient phase is 10.3 s−1, which is considerably faster than the turnover number of the ATPase reaction (0.011 s−1). A similar burst, equivalent to 0.57 mol of enzyme, is observed in the time course of the biosynthetic reaction at 10 °C, in which the transient rate constant of 88 s−1 is also much faster than the turnover number of the biosynthetic reaction (4.0 s−1, 10 °C). When these data are combined with the results of positional isotope exchange of [γ-18O]ATP [Midelfort, C.F., & Rose, I.A. (1976) J. Biol. Chem. 251, 5881–5887], it is shown that the enzyme-bound intermediate responsible for the burst of acid-labile phosphate is γ-glutamyl phosphate. In the biosynthetic reaction pathway, γ-glutamyl phosphate is formed after NH4+ binds to the enzyme, and the presence of NH4+ in this quaternary E-MgATP-Glu-NH4+ complex effects an increase in the rate of phosphoryl group transfer that is substantially faster than that of the E-MgATP-Glu complex in the ATPase reaction. Because it is formed and consumed more rapidly than the net rate of catalysis, γ-glutamyl phosphate is a kinetically competent intermediate of both reaction pathways. At 10 °C, an identical transient phase (rate constant = 96 s−1) is observed by measuring changes in protein fluorescence intensity with the stopped-flow technique, which indicates that the chemical formation of γ-glutamyl phosphate is accompanied by a change in enzyme conformation. From isotope partitioning experiments, it is found that MgATP is “sticky” in the binary E-MgATP complex (koff = 0.2V1/Et = 2.9 s−1) and is infinitely sticky in the E-MgATP-Glu and E-MgATP-Glu-NH4+ complexes. Since the rate of MgATP release from E-MgATP is nearly twice the turnover number of the reverse biosynthetic reaction (V2/Et = 1.65 s−1), MgATP release is not solely rate limiting for the reverse reaction. Glutamate dissociates from E-MgATP-Glu at a rate of 19 s−1 (1.3V1/Et) and is infinitely sticky in the E-MgATP-Glu-NH4+ complex. Glutamate dissociates so rapidly from the E-Glu complex as to render it insignificant to the biosynthetic reaction pathway. These results are in accord with a kinetic mechanism that is predominantly ordered [Meek, T.D., & Villafranca, J.J. (1980) Biochemistry 19, 5513–5519]. From the results of isotope partitioning in the reverse reaction, it is shown that the rate-limiting step of the forward biosynthetic reaction is the release of MgADP from E-MgADP. The rate constant for this dissociation is 14 s−1, which equals V1/E1 and is 8 times the value of V2/Et. © 1982, American Chemical Society. All rights reserved.

author list (cited authors)

  • Meek, T. D., Johnson, K. A., & Villafranca, J. J.

citation count

  • 50

publication date

  • April 1982