Sandbox Reserved 764: Difference between revisions

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==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 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 within this cleft 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 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.

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 ==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 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 within this cleft 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 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.