Glutamine synthetase kinetics

RIBULOSE-5-PHOSPHATE 3-EPIM ERASE Escherichia coll glutamine synthetase, ENZYME CASCADE KINETICS GLUTAMINE SYNTHETASE Essential amino acid residues in catalysis, AFFINITY LABELING ESTERASES... 

The kinetic reaction mechanism appears to be random, and for the reaction to proceed, all substrates must reside as a E-D-Ala-D-Ala-MgATP quaternary complex. Except for its activation of an a-carboxylate to form a peptide bond, the enzyme s mechanism appears to be completely analogous to that catalyzed by glutamine synthetase, which forms a y-glutamyl-phosphate intermediate. There is strong evidence for the participation... 

Since the reaction catalyzed by glutamine synthetase has three substrates (L-glutamate, NH3, and metal-ATP) and three products (L-glutamine, P , and metal-ADP), the kinetic mechanism as deduced by steady-state kinetics... 

Fig. 19. Two proposed kinetic schemes for the glutamine synthetase reaction.

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 ... 

Butterfield DA, Drake J, Pocemich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer s disease brain central role for amyloid beta-peptide. Trends Mol Med 7 548-554 Butterfield DA, Hensley K, Htirris M, Mattson M, Carney J (1994) beta-Amyloid peptide free radical fragments initiate synaptosomal Upoperoxidation in a sequence-specific fashion impUcations to Alzheimer s disease. Biochem Biophys Res Commun 2(X) 710-715 Butterfield DA, Hensley K, Cole P, Subramaniam R, Aksenov M, Aksenova M, Bummer PM, Haley BE, Carney JM (1997) Oxidatively-induced structural alteration of glutamine synthetase assessed by analysis of spin labeled incorporation kinetics relevance to Alzheimer s disease. J Neurochem 68 2451-2457... 

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 PIX experiment has been extensively applied to problems of the type presented by glutamine synthetase, where the putative intermediates are too unstable to study directly and information on their presence and rates of formation is needed. For tests of kinetic competence this technique can be superior to more direct experiments, in which isolated or synthesized intermediates are mixed with enzymes in order to construct active complexes and the rates of reactions are measured. In such experiments the rates are rarely if ever found to be kinet-ically competent, since the putative intermediate must bind to an enzyme form that is never generated in a catalytic cycle, and the rate at which the intermediate complex is formed will be less than the overall rate. In the PIX experiment only the normal catalytic reaction is used to generate the desired complexes. The main drawback to the PIX experiment is that it depends on the torion rate in an intermediate such as MgADP. When the torsion rate is slow, e.g., owing to metal complexation, the experiment fails. 

Care should be taken when determining the kinetics of the ammonia-dependent reaction that glutamine synthetase is not present in the extracts. Glutamine could easily be formed from glutamate and ammonia in the presence of ATP and Mg and act as the substrate for the AS reaction being assayed. 

In considering the commercially successful herbicides known to be inhibitors of amino acid biosynthesis, three aspects will be considered the kinetic description of the inhibition, the molecular interactions involved, and the relevance of the proposed target site to the observed physiological effects. Because of the extensive body of research published on the shikimate pathway and the herbicide glyphosate, this example will be taken as a paradigm of the inhibitor-enzyme relationship. The other cases considered will be the branched-chain amino acid family, histidine biosynthesis, and glutamine synthetase. 

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