Determination of the mechanism of the argininosuccinate synthetase reaction by static and dynamic quench experiments.
The reactions catalyzed by argininosuccinate synthetase have been examined by the use of static and dynamic quench techniques. The time course of the forward reaction (22 degrees C) at pH 8.0 is characterized by a "burst" of AMP formation upon quenching with acid that is equivalent to 0.59 mol of enzyme. The pre-steady-state rate is followed by a slower steady-state rate of 0.60 s-1. The rate constant for the transient phase is 9.7 s-1. The time course for the formation of argininosuccinate is linear and shows neither a "lag" nor a burst phase. These results have been interpreted to mean that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. In the presence of aspartate the rate constant for the formation of citrulline adenylate (6.2 s-1) from ATP and citrulline is 7 times faster than the rate of formation of argininosuccinate from aspartate and citrulline adenylate (0.9 s-1). This suggests that the second step is predominantly rate limiting. The rate constant for the formation of citrulline adenylate in the absence of enzyme-bound aspartate (0.01 s-1) is 600 times slower than when aspartate is present. This indicates that the binding of aspartate to the enzyme regulates the formation of the intermediate. These results are in complete accord with our previously published steady-state kinetic scheme showing sequential addition of substrates.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001124:Argininosuccinate Synthase / Q000378:metabolism*; D002417:Cattle; D002956:Citrulline / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008433:Mathematics
D002956:Citrulline; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D001124:Argininosuccinate Synthase