Glutamine synthetase structure

X-ray crystal structures of glutamine synthetase from both Salmonella typhimuriuni and Mycobacterium tuberculosis are very similar. Structures of wild type enzymes and of active site mutants have been determined. All structures have been solved with Mn in the active site. There are twelve identical subunits arranged in two face-to-face symmetrical hexamers. The active sites are in funnel-shaped open-ended cavities located between adjacent subunits of the hexamer. These cavities are 45 A long, 30 A wide at the outer end, and 10 A wide at the inner end and the active site with the two Mn " ions is approximately halfway down the cavity. The metal-metal distance is 5.8 A. The more tightly bound Mn is coordinated to the side chains of Glu-131, Glu-212, Glu-220, and two water molecules, one of which is shared by both metal ions. Glu-129, Glu-357, His-269, and two additional water molecules are bound to the Mn + at the lower affinity site. A schematic view of the active site metal coordination is shown in Figure 36. 

FIGURE 22-5 Subunit structure of glutamine synthetase as determined by x-ray diffraction. (PDB ID 2GLS) (a) Side view. The 12 subunits are identical they are differently colored to illustrate packing and placement, (b) Top view, showing active sites (green). 

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. 

Many years ago Meister s group (96-101) synthesized of a number of inhibitors or substrate analogs that would mimic the intermediates in the proposed pathway of the reactions catalyzed by glutamine synthetase. Two of these are listed below, along with the structures for substrates and two alleged intermediates. 

The above data, along with other data from Meister s group (102, 103), provided the basis for computer modeling studies (104) of the active site conformations of substrates and intermediates in the glutamine synthetase reaction. The conclusions are that both structures II and III are in the reaction mechanism. 

Our studies of the substrates [glutamate (I) and ATP] and of substrate analogs [AMP-P-(CH2)-P and methionine sulfoximine] reveal interactions between both substrate sites and both metal ion sites. Previously mentioned studies by Meister s group showed that the irreversible inhibition of glutamine synthetase in the presence of L-methionine (S)-sulfoximine and ATP was due to formation of the sulfoximine phosphate (IV). The tetrahedral geometry at the sulfur atom of the sulfoximine was suggested to be a mimic of the active structure of the adduct of y-glutamyl phosphate and ammonia (III). Data in our laboratory provide spectroscopic evidence that methionine... 

This section summarizes recent data obtained from biophysical techniques on the nature of the metal ion, catalytic, and modifier sites of E. coli glutamine synthetase. Distance relationships between these sites are given in Fig. 27. This enzyme has many diverse modes of biochemical control. Recent data presented in this review demonstrate that progress is being made toward understanding the structural basis for the control of enzymic catalysis. When x-ray data become available 127), the amino acid groups at the catalytic site will be known and will provide further details concerning the mechanism of the enzymic reaction. 

There are two cation-binding sites per subunit, classified nL and n2. The n, site is a structural site, which may involve the reorientation of a glutamate carboxyl group. Metals bind first at this site. The n2 site is the catalytic site and may bind metal or metal-nucleotide. Co111 and Cr111 can be incorporated into the nj metal-binding sites in un-adenylylated glutamine synthetase from E. co/i.318 Both derivatives were inactive, but were able to bind Mn2+ at the n2 site. Comparison of the quaternary enzyme-Crin-Mnn-ADP (which shows spin-spin interaction between the two metal centres) with enzyme-Com-Mnn-ADP leads to an estimate of the distance between n, and n2 sites of 7 2 A. 

Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. The EMBO Journal 5, 1429-35. 

Gebhardt, C., Olivier, J.E., Forde, B.G., Saarelainen, R. Miflin, B.J. (1986). Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. The EMBO Journal 5, 1429-35. 

D Eisenberg, HS Gill, GM Pfluegl, SH Rotstein (2000) Structure-function relationships of glutamine synthetases, Biochim Biophys Acta 1477(1) 122—145... 

RJ Almassy, CA Janson, R Hamlin, NH Xuong, D Eisenberg (1986) Novel subunit-subunit interactions in the structure of glutamine synthetase, Nature 323(6086) 304-309... 

S H Liaw, D Eisenberg (1994) Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substtate complexes, Biochemistry 33(3) 675—681... 

H S Gill, D Eisenberg (2001) The Crystal Structure of Phosphinothricin in the Active Site of Glutamine Synthetase Illuminates the Mechanism of Enzymatic Inhibition, Biochemistry 40(7) 1903-1912... 

Figure II-4 Examples of the quaternary structure of proteins, (a) A drawing of glutamine synthetase of coli showing the orientation of the 12 identical subunits of the enzyme. (b) A drawing of aspartate transcarbamylase of coli showing the proposed orientation of the 6 catalytic subunits (labeled C, each MW = 33,000), and 6 regulatory subunits (labeled R, each MW =...

Norenberg, M.D., and A.Martinez-Hernandez. 1979. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161(2) 303—310. 

The Glutamine Synthetase of Escherichia coli Structure and Control... 

Figure 3. Comparison of structures of several compounds with potent iQ vitro glutamine synthetase-inhibiting properties.

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... 

Kramer, J. G., Wyman, M., Zehr, J. P., and Capone, D. G. (1996). Diel variability in transcription of the structural gene for glutamine synthetase (gin A) in natural populations of the marine diazotrophic cyanobacterium Trichodesmium thiebautii. FEMS Microbiol. Ecol. 21, 187-196. 

Figure 24.25. Structure of Glutamine Synthetase. Glutamine synthetase consists of 12 identical subunits arranged in two rings of six subunits. The active sites are indicated by the presence of manganese ions (two yellow spheres).

Figure 24.27. Structure of the Regulatory Protein P. This trimeric regulatory protein controls the modification of glutamine synthetase. 

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