Isotope exchange synthetase

P Aminoacyl-tRNA synthetases Detection of intermediates by quenched flow, steady state kinetics, and isotope exchange... 

The kinetic approach that has been most extensively applied to glutamine synthetase is that of equilibrium isotope exchange (85-88). Experimental procedures involve preparing a series of reaction solutions at chemical equilibrium and monitoring the following exchanges ... 

Recently Midelfort and Rose (4) introduced a positional isotope exchange technique that is also suitable for study by the chemical shift technique. Briefly, this technique follows the exchange of label from one part of a substrate to another due to rotational equivalence of some intermediate. The method was first applied to glutamine synthetase in the reaction ... 

The positional Isotope exchange has also been measured with 31p-nmr In the reverse reaction of carbamyl phosphate synthetase ... 

There are several remarkable features of this isotope-exchange assay. If the reaction progress is followed, equilibrium of isotope exchange is attained at a defined distribution of the label between ATP and PPi. This has been shown in the case of ACV synthetase by Baldwin and colleagues [57], but is rarely done because of the restrictions of enzyme stability. Evaluation of this equilibrium, which has been reached in the case of the A. chrysogenum enzyme in about 3 hr, has never been attempted, but the concentrations roughly reflect the initial rates of the exchange reactions. 

To examine the kinetic competence of y-glutamyl phosphate formation at the active site of glutamine synthetase, the technique of positional isotope exchange (PIX) was introduced to studies of the mechanism of phospho transfer (85). This experiment circumvented the problem of the instability of the intermediate, which complicated direct kinetic experiments. In the PIX experiment, the rate at which glutamine synthetase catalyzes reaction (25) was measured. 

The positional isotopic exchange (PIX) technique involves observation of exchange of label between two positions in a reactant. The method was first used by Midelfort and Rose in 1976 to follow jS-y bridge to )8-nonbridge transfer of 0 in ATP incubated with glutamine synthetase (60) ... 

With respect to mechanism of action, the most extensive kinetic and equilibrium exchange studies have been carried out on monofunctional 10-formyl-H4-folate synthetase from Cl. cylindrosporum [84]. The data support a random sequential mechanism that does not involve the formation of freely dissociable intermediates. The most likely mechanism, however, is not concerted but probably involves the formation of a formyl phosphate intermediate, since the synthetase catalyzes phosphate transfer from carbamyl phosphate but not acetyl phosphate to ADP with H 4-folate serving as an activator. Carbamyl phosphate is an inhibitor of 10-formyl-H 4-folate synthesis - an inhibition that can be eliminated only when both ATP and formate are present in accord with the concept that it spans both sites [85]. It would be of considerable interest to attempt to demonstrate positional isotope exchange employing [, y- 0]ATP for this enzyme in order to further implicate an enzyme-bound formyl phosphate species [86]. 

One of the most useful appKcations of isotope exchange studies comes in determining the order of addition of substrates in trisubstrate Ping Pong mechanisms. The best way to understand this problem is in relation to a specific example. Consider, for example, the reaction catalyzed by a large number of synthetases (Walsh, 1998) ... 

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