Active site glutamine synthetase

Many enzymes (see Chapters 14 to 16) derive at least some of their catalytic power from oligomeric associations of monomer subunits. This can happen in several ways. The monomer may not constitute a complete enzyme active site. Formation of the oligomer may bring ail the necessary catalytic groups together to form an active enzyme. For example, the active sites of bacterial glutamine synthetase are formed from pairs of adjacent subunits. The dissociated monomers are inactive. 

L-Glutamate, glutamine synthetase intermediate, 28 356, 361, 365 Glutamine synthetase, 28 349-366 active site, 28 350... 

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). 

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. 

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. 

As discussed earlier, the enzymic reaction catalyzed by glutamine synthetase requires the presence of divalent metal ions. Extensive work has been conducted on the binding of Mn2+ to the enzyme isolated from E. coli (82, 109-112). Three types of sites, each with different affinities for Mn2+, exist per dodecamer n, (12 sites, 1 per subunit) of high affinity, responsible for inducing a change from a relaxed metal ion free protein to a conformationally tightened catalytically active protein n2 (12 sites) of moderate affinity, involved in active site activation via a metal-ATP complex and n3 (48 sites) of low affinity unnecessary for catalysis, but perhaps involved in overall enzyme stability. The state of adenylylation and pH value alter the metal ion specificity and affinities. 

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... 

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). 

The main role of the IIA cations in the activation of enzymes seems to be that of weak Lewis acids. In addition the cation may serve as a template to bridge enzyme and substrate and bring them into the correct relative orientation for reaction. Furthermore these cations may stabilize or produce certain protein conformations. Thus glutamine synthetase binds 24 moles of Mn2+ per mole of protein. The binding of the first 12 cations results in conformational changes that lead to the formation of 12 new sites for the binding of the remaining 12 cations, which then have a catalytic role. 

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 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 25.5. Substrate Chanueliug. The three active sites of carbamoyl phosphate synthetase are linked by a channel (yellow) through which intermediates pass. Glutamine enters one active site, and carbamoyl phosphate, which includes the nitrogen atom from the glutamine side chain, leaves another 80 A away.

Mg -specific site into an Mn -specific site, with decrease in activity. Glutamine synthetase from... 

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