Escherichia coli glutamine synthetase. Determination of rate-limiting steps by rapid-quench and isotope partitioning experiments.
Academic Article
Overview
Research
Identity
Additional Document Info
Other
View All
Overview
abstract
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 s1, which is considerably faster than the turnover number of the ATPase reaction (0.011 s1). 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 s1 is also much faster than the turnover number of the biosynthetic reaction (4.0 s1, 10 C). When these data are combined with the results of positional isotope exchange of [-18O]ATP [Midelfort, C.F., & Rose, I.A. (1976) J. Biol. Chem. 251, 58815887], it is shown that the enzyme-bound intermediate responsible for the burst of acid-labile phosphate is -glutamyl phosphate. In the biosynthetic reaction pathway, -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, -glutamyl phosphate is a kinetically competent intermediate of both reaction pathways. At 10 C, an identical transient phase (rate constant = 96 s1) is observed by measuring changes in protein fluorescence intensity with the stopped-flow technique, which indicates that the chemical formation of -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 s1) 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 s1), MgATP release is not solely rate limiting for the reverse reaction. Glutamate dissociates from E-MgATP-Glu at a rate of 19 s1 (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, 55135519]. 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 s1, which equals V1/E1 and is 8 times the value of V2/Et. 1982, American Chemical Society. All rights reserved.
