Sandbox Reserved 764: Difference between revisions

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The three-dimensional structure of EPSP synthase in ''Escherichia coli'' was determined using X-ray crystallography. The enzyme has two distinct domains, 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.

~~<scene name='56/564040/Secondary_elements/2'>bound</scene>~~

~~<scene name='56/564040/Secondary2/1'>unbound</scene>~~

[[Image:axisofsym.jpg|left|frame|Figure 3. Illustration of...]]

==EPSP Synthase Mechanism==

In order to catalyze the reversible transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), EPSP synthase must bind both S3P and PEP. Fluorescence studies show that both S3P and PEP are capable of binding to free EPSP synthase <ref>PMID:3052285</ref>. However, it has been found that PEP preferentially interacts with the E-S3P complex <ref>PMID:3052285</ref>. Steady-state and pre-steady-state kinetic studies indicate that the kinetically preferred reactions pathway occurs with S3P binding to EPSP synthase first, followed by PEP <ref>PMID:10353849</ref>. Prior to the binding of any substrate, EPSP synthase exists in an <scene name='56/564040/Secondary2/1'>unbound</scene> open conformation. In this conformation, the domains are hinged apart in such a way that they create a cleft or well of cationic charges that draw the anionic substrates into the active site. After S3P and PEP ~~BOUND~~ to EPSP synthase, the domains undergo a conformational change into a closed formation. Chemical modification studies on EPSPS indicate that Lysine, Arginine, and Histidine residues are essential for activity of the enzyme. The reactive Lysine and Arginine residues have been identified as <scene name='56/564040/Lys22/4'>Lys-22</scene> and <scene name='56/564040/Arg27/3'>Arg-27</scene>. 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>. It is speculated that both Lys-22 and Arg-27 constitute a part of the active site recognizing the anionic residues on the substrates. The following residues were proposed to be involved in the enzyme mechanism:

In order to catalyze the reversible transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), EPSP synthase must bind both S3P and PEP. Fluorescence studies show that both S3P and PEP are capable of binding to free EPSP synthase <ref>PMID:3052285</ref>. However, it has been found that PEP preferentially interacts with the E-S3P complex <ref>PMID:3052285</ref>. Steady-state and pre-steady-state kinetic studies indicate that the kinetically preferred reactions pathway occurs with S3P binding to EPSP synthase first, followed by PEP <ref>PMID:10353849</ref>. Prior to the binding of any substrate, EPSP synthase exists in an <scene name='56/564040/Secondary2/1'>unbound</scene> open conformation. In this conformation, the domains are hinged apart in such a way that they create a cleft or well of cationic charges that draw the anionic substrates into the active site. After S3P and PEP are <scene name='56/564040/Secondary_elements/2'>bound</scene> to EPSP synthase, the domains undergo a conformational change into a closed formation. Chemical modification studies on EPSPS indicate that Lysine, Arginine, and Histidine residues are essential for activity of the enzyme. The reactive Lysine and Arginine residues have been identified as <scene name='56/564040/Lys22/4'>Lys-22</scene> and <scene name='56/564040/Arg27/3'>Arg-27</scene>. 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>. It is speculated that both Lys-22 and Arg-27 constitute a part of the active site recognizing the anionic residues on the substrates. 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.

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 The three-dimensional structure of EPSP synthase in ''Escherichia coli'' was determined using X-ray crystallography. The enzyme has two distinct domains, 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.
-<scene name='56/564040/Secondary_elements/2'>bound</scene>
-<scene name='56/564040/Secondary2/1'>unbound</scene>
 [[Image:axisofsym.jpg|left|frame|Figure 3. Illustration of...]]
 ==EPSP Synthase Mechanism==
-In order to catalyze the reversible transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), EPSP synthase must bind both S3P and PEP. Fluorescence studies show that both S3P and PEP are capable of binding to free EPSP synthase <ref>PMID:3052285</ref>. However, it has been found that PEP preferentially interacts with the E-S3P complex <ref>PMID:3052285</ref>. Steady-state and pre-steady-state kinetic studies indicate that the kinetically preferred reactions pathway occurs with S3P binding to EPSP synthase first, followed by PEP <ref>PMID:10353849</ref>. Prior to the binding of any substrate, EPSP synthase exists in an <scene name='56/564040/Secondary2/1'>unbound</scene> open conformation. In this conformation, the domains are hinged apart in such a way that they create a cleft or well of cationic charges that draw the anionic substrates into the active site. After S3P and PEP BOUND to EPSP synthase, the domains undergo a conformational change into a closed formation. Chemical modification studies on EPSPS indicate that Lysine, Arginine, and Histidine residues are essential for activity of the enzyme. The reactive Lysine and Arginine residues have been identified as <scene name='56/564040/Lys22/4'>Lys-22</scene> and <scene name='56/564040/Arg27/3'>Arg-27</scene>. 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>.  It is speculated that both Lys-22 and Arg-27 constitute a part of the active site recognizing the anionic residues on the substrates. The following residues were proposed to be involved in the enzyme mechanism:
+In order to catalyze the reversible transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), EPSP synthase must bind both S3P and PEP. Fluorescence studies show that both S3P and PEP are capable of binding to free EPSP synthase <ref>PMID:3052285</ref>. However, it has been found that PEP preferentially interacts with the E-S3P complex <ref>PMID:3052285</ref>. Steady-state and pre-steady-state kinetic studies indicate that the kinetically preferred reactions pathway occurs with S3P binding to EPSP synthase first, followed by PEP <ref>PMID:10353849</ref>. Prior to the binding of any substrate, EPSP synthase exists in an <scene name='56/564040/Secondary2/1'>unbound</scene> open conformation. In this conformation, the domains are hinged apart in such a way that they create a cleft or well of cationic charges that draw the anionic substrates into the active site. After S3P and PEP are <scene name='56/564040/Secondary_elements/2'>bound</scene> to EPSP synthase, the domains undergo a conformational change into a closed formation. Chemical modification studies on EPSPS indicate that Lysine, Arginine, and Histidine residues are essential for activity of the enzyme. The reactive Lysine and Arginine residues have been identified as <scene name='56/564040/Lys22/4'>Lys-22</scene> and <scene name='56/564040/Arg27/3'>Arg-27</scene>. 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>.  It is speculated that both Lys-22 and Arg-27 constitute a part of the active site recognizing the anionic residues on the substrates. 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.