Determination of rate-limiting steps of Escherichia coli carbamoyl-phosphate synthase. Rapid quench and isotope partitioning experiments.
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The ATPase and carbamoyl phosphate synthesis reactions of Escherichia coli carbamoyl-phosphate synthase have been examined by using rapid quench and isotope partitioning techniques. The time course of the ATPase reaction is characterized by a burst of 1 enzyme equiv of acid-labile phosphate, followed by a slower steady-state rate (0.20 s-1). The rate constant for the transient phase (4.5 s-1) is close to the overall rate constant for the carbamoyl phosphate synthesis reaction, suggesting that the formation of carboxy phosphate is rate limiting for the overall reaction (3.1 s-1). Binding of both molecules of ATP prior to the addition of glutamine could not be demonstrated by using isotope partitioning experiments. This suggests that either the ATP used in the reaction for the phosphorylation of carbamate does not bind significantly to the enzyme until after the addition of glutamine or its dissociation rate constant from the enzyme must be very fast compared with the rate constant for the overall reaction. In addition, carbamoyl-phosphate synthase was found not to catalyze an 18O exchange from [7-18O]ATP during the ATPase reaction. When carbamoyl-phosphate synthase was incubated with 32Pi, carbamoyl phosphate, and ADP, less than 1% of the ATP synthesized resulted from reaction with the Pi This suggests that the complete forward reaction cannot be totally reversed and that some intermediate (presumably carbamate) must be dissociating irreversibly from the enzyme at least 100 times faster than its reaction with P, All of the above data are in accord with our previously published steady-state kinetic scheme showing sequential addition of substrates [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. 1979, American Chemical Society. All rights reserved.
author list (cited authors)
Raushel, F. M., & Villafranca, J. J.
complete list of authors
Raushel, FM||Villafranca, JJ