Sandbox Reserved 764: Difference between revisions

The three-dimensional structure of EPSP synthase in ''Escherichia coli'' was determined using X-ray crystallography. The enzyme has two distinct <scene name='56/564040/Two_domains/1'>domains</scene>, each of which have a radius of approximately 25 Angstroms <ref>PMID:11607190</ref>. The two domains are joined together by two crossover chain segments with both the amino and carboxyl terminus of the polypeptide chain located in the C-terminal domain. In its entirety, EPSP synthase is composed of the following <scene name='56/564040/Epsp_synthase/2'>secondary structures</scene>: twelve alpha helices and twenty-four beta sheets. The domains are formed by protein folding subunits consisting of two parallel helices and  four beta sheets <ref>PMID:11607190</ref>. Each domain is formed from three of these subunits, forming the threefold axis of symmetry shown below in Figure 3. Three alpha helices are positioned around the axis of each domain to form its core. The domain surfaces consist of three beta-sheets and three parallel helices that are all solvent accessible <ref>PMID:11607190</ref>. The beta sheets can be found in both the parallel and anti-parallel positions. The alpha helices are positioned in a parallel fashion that establishes equivalent chain polarities throughout the enzyme. It is believed that the helix dipole establishes optimal interaction between the enzyme and charged phosphate groups of the substrates <ref> W. G. J. Hol, P. T. van Duijnen & H. J. C. The [alpha]-helix dipole and the properties of proteins: Nature 273, 443-446 (8 June 1978).[http://dx.doi.org/10.1038/273443a0 DOI:10.1038/273443a0</ref>. Studies suggest that these helical dipole efffects create a cationic well that draws the anionic substrates into the active site located in the interdomain cleft.  

Susanne Eschenburg, Wendy A. Shuttleworth, John V. Schloss, Nikolaus Amrhein, Jeremy N. S. Evans, Wolfgang Kabsch. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail : PNAS 98, 1376-1380 (13 February 2001).[DOI:10.1073/pnas.98.4.1376] </ref>. After S3P and PEP are bound to EPSP synthase, the domains undergo a conformational change into a <scene name='56/564040/Secondary_elements/2'>closed</scene> formation. Chemical modification studies on EPSPS indicate that Lysine, Arginine, and Histidine residues within this cleft are essential for activity of the enzyme. The reactive Lysine and Arginine residues in this cleft have been identified as <scene name='56/564040/Lys22/4'>Lys-22</scene> and <scene name='56/564040/Arg27/3'>Arg-27</scene>. Studies suggest that both Lys-22 and Arg-27 constitute a part of the active site recognizing the anionic residues on the substrates.Mutation of Lys-22 to Arg does not affect the activity of the enzyme. However, mutation of Lys-22 to either Ala or Glu leads to complete loss of enzyme activity; therefore suggesting that the cationic nature of the Lysine side chain plays an important role in the activity of EPSP synthase <ref>PMID:3052285</ref>. The following residues were proposed to be involved in the enzyme mechanism:

-E341 may as a proton donor for the C-3 of PEP. In the x-ray structure, E341 is located close to the C-2 position of PEP, and thus the carboxyl oxygen of E341 could stabilize PEP oxonium ion.  

-His-385 of which is only 2.85 Å away from O of E341 may function as proton source for E341.  

-Two residues, D313 and K22 may act as a proton acceptor of the 5-OH of S3P which should be deprotonated to attack the C-2 of PEP (24) and interacts only with those two residues (Scheme 1).  

-R100, D242, and D384 residues are not involved in substrate binding/catalysis, but mutagenisis results show that they are required. It is proposed that they may play a role in domain closure or stabilize the closed conformation.