Sandbox Reserved 763: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 107: | Line 107: | ||
Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116. The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One. | Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. Shikimate binding occurs with helices α2, α3, and α4 (N-terminal region).<ref name="3dstructure" /> The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116. The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One. | ||
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394/#!po=19.5652</ref> | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394/#!po=19.5652</ref> | ||
===Protein Fold=== | ===Protein Fold=== | ||
| Line 141: | Line 113: | ||
Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi | Shikimate kinase is a convenient protein to use in protein folding studies. This is because it is one of the smallest kinases and is a monomeric enzyme. It has been shown that the proposed refolding model includes a rapid hydrophobic collapse and then a slower secondary structure formation.<ref name="fold">The refolding of type II shikimate kinase from Erwinia chrysanthemi | ||
after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref> | after denaturation in urea. European Journal of Biochemistry http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.ejb.02862.x/pdf</ref> | ||
===Methods Used to Solve/Study=== | ===Methods Used to Solve/Study=== | ||
| Line 169: | Line 140: | ||
Isothermal titration calorimetry | Isothermal titration calorimetry | ||
==Mechanism of Action== | ==Mechanism of Action== | ||
| Line 176: | Line 145: | ||
===Reaction Pathway=== | ===Reaction Pathway=== | ||
SK is found in the shikimate pathway, which is present in bacteria, fungi, higher plants, algae, and apicomplexan. This pathway serves to convert erythrose-4-phosphate into chorismate. Chorismate is a required intermediate in the biosynthesis of aromatic amino acids and secondary metabolites. Shikimate kinase is the catalyst for the fifth step of the shikimate pathway, where shikimate (SKM) is converted into shikimate-3-phosphate (S3P) and ATP serves as a co-substrate. It has been found experimentally that SK and shikimate binding involves two steps: substrate attaches to binding site in the first step and LID closure occurs in the second step.<ref name="miscinfo" /> | SK is found in the shikimate pathway, which is present in bacteria, fungi, higher plants, algae, and apicomplexan. This pathway serves to convert erythrose-4-phosphate into chorismate. Chorismate is a required intermediate in the biosynthesis of aromatic amino acids and secondary metabolites. Shikimate kinase is the catalyst for the fifth step of the shikimate pathway, where shikimate (SKM) is converted into shikimate-3-phosphate (S3P) and ATP serves as a co-substrate. It has been found experimentally that SK and shikimate binding involves two steps: substrate attaches to binding site in the first step and LID closure occurs in the second step.<ref name="miscinfo" /> As seen to the left, SK catalyzes the transfer of a phosphate group from from ATP to the 3-hydroxyl group of shikimate. This reaction is reversible, though the forward reaction forming S3P is the important reaction in chorismate synthesis. | ||
===Ligands=== | |||
The liganded state of SK includes binary and ternary complexes. | |||
Examples of ligands include: | |||
<scene name='56/564039/Skmligand/1'>SKM</scene> | |||
<scene name='56/564039/Skm3po4ligand/1'>shikimate 3-phosphate</scene> | |||
<scene name='56/564039/Adpligand/1'>ADP</scene> | |||
<scene name='56/564039/Mgadpligand/1'>MgADP</scene> | |||
<scene name='56/564039/Amppcpligand/1'>AMPPCP</scene> | |||
<scene name='56/564039/Amppnpligand/1'>AMPPNP</scene> | |||
<scene name='56/564039/Atpligand/1'>ATP</scene> | |||
<scene name='56/564039/Clligand/2'>Chloride ion</scene> | |||
=== | <scene name='56/564039/Sulfate/1'>Sulfate</scene> | ||
Magnesium ions bind in the active site and hold influence over the position of shikimate hydroxy groups. This ion has a role in the transfer of the γ-phosphate of ATP to the 3-hydroxy group on shikimate. Chloride ions increase enzyme affinity for ADP and ATP and help to bind the nucleotide substrate in correct orientation.<ref name="mgcl"> Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis Structural Biology and Crystallization Communications | |||
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330112/</ref> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> Sulfate interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" /> | |||
===Inhibitors=== | ===Inhibitors=== | ||
