Fluorescence glutamine synthetase

Escherichia coli have also developed an elegant method to control enzyme catalysis that occurs by covalent modification of each subunit. In this latter reaction a single tyrosyl residue per subunit is adenylylated to produce a stable 5 -adenylyl-O-tyrosyl derivative. Recent NMR and fluorescence data will be reviewed concerning the nature of this adenylyl site and its spatial relationship to the metal ions at the catalytic site. The enzymes responsible for the covalent adenylylation reaction comprise a cascade system for amplifying the activation or inactivation of glutamine synthetase molecules (81). 

Experimental techniques for determining distances must be employed to establish the structure of the active site components of glutamine synthetase. Techniques that are available for these studies are x-ray crystallography, EPR, NMR, and fluorescence energy transfer. All approaches are currently being employed to study the structure and function of this metalloenzyme. 

Three physical methods, vis., NMR, EPR, and fluorescence energy transfer, were used to determine the spatial relationship between the adenylyl, catalytic ( 2), and the divalent metal ion activating sites (n ). Glutamine synthetase of low adenylylation state ( 10) was enzymatically adenylylated with either [2-13C] ATP, e-ATP(l-A -etheno-ATP), or 6-amino-TEMPO-ATP (121,122). 

Fig. 26. Summary of [13C], [31F]-NMR data, fluorescence energy transfer data and EPR data on various derivatives at the covalent adenylyl site on glutamine synthetase. Reprinted with permission of Academic Press from Villafranca et al. (115).

Figure 26 also depicts the distances calculated from fluorescence quenching experiments by enzyme-bound Co2+ on the s-adenosine moiety of s-ATP adenylylated glutamine synthetase (e-AMP-GS). Additional data were gathered by an EPR method that measured the decrease in EPR amplitude of the nitroxide spin-labeled adenylylated enzyme (TEMPO-AMP-GS) owing to enzyme-bound Mn2+ (122). Distances between Mn2+ and the N—O of the spin label are also shown in Fig. 26. 

Thus, we have determined the distances between the adenylyl moiety and the two divalent metal ion binding sites on glutamine synthetase by 13C and 3 P NMR, spin-labeled EPR, and fluorescence energy transfer methods. The results obtained from each method are in good agreement. The data show that the adenylyl regulatory site is close to the catalytic site (12-20 A). Additional data on the rotational correlation time of the adenyl derivatives reveal that the adenylyl site is located on the surface of the enzyme. 

Some plant pathogenic bacteria and their phytotoxins have been screened in bioassays that monitor the effects of their toxins (antibiotic and phytotoxic) on other sensitive bacteria. For example, several fluorescent Pseudomonas syringae pvs. produce extracellular phytotoxins.76,106,116 Tabtoxin is produced by P. syringae pv. tabaci and pv. coronafacines, and this natural product inhibits glutamine synthetase.34,46,116 Phaseolotoxin, produced by P. syringae pv. phaseolicola... 

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