Sandbox Reserved 1068: Difference between revisions

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'''Isochorismate synthase (IS)'''

Currently, isochorismate is believed to be formed from chorismate through a proposed Sn2 mechanism involving nucleophilic attack of an activated water molecule to the C2 center followed by either a concerted or stepwise elimination of the C4 hydroxyl group (Figure 7)<ref name="9a"/><ref name="6a"/><ref name="8a"/>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons<ref name="6a"/><ref name="8a"/>. However, mutational analysis of Lys205 suggested that the lysine reside is not the sole determinant in the activation of a water molecule for nucleophilic attack of the C2 center. Studies have shown that Lys205 is protonated at neutral pH and therefore can't act as a base to activate the water molecule, agreeing with the mutational analysis data. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule. The magnesium ion forces the negatively charged Glu297 residue to face toward the active site and the pKa of Glu297 (3.9) suggest an unprotonated state. Furthermore, Glu297 forms a hydrogen bond with a water molecule within the active site as well as with Lys205, which is in turn hydrogen bonded to C1 carboxylate group of chorismate and the oxygen of the nucleophilic water molecule. The glutamic residue (Gly252) could protonate the C4 leaving hydroxyl group. The pKa of Gly252 (7.7) suggest that is it is the only protonated glutamate residue in the active site at pH 7 and thus able to protonate the C4 leaving group. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5.

Currently, isochorismate is believed to be formed from chorismate through a proposed Sn2 mechanism involving nucleophilic attack of an activated water molecule to the C2 center followed by either a concerted or stepwise elimination of the C4 hydroxyl group (Figure 7)<ref name="9a"/><ref name="6a"/><ref name="8a"/>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons<ref name="6a"/><ref name="8a"/>. However, mutational analysis of Lys205 suggested that the lysine reside is not the sole determinant in the activation of a water molecule for nucleophilic attack of the C2 center<ref name="6a"/><ref name="8a"/>. Studies have shown that Lys205 is protonated at neutral pH and therefore can't act as a base to activate the water molecule, agreeing with the mutational analysis data<ref name="1a"/><ref name="2a"/>. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule<ref name="6a"/><ref name="8a"/>. The magnesium ion forces the negatively charged Glu297 residue to face toward the active site and the pKa of Glu297 (3.9) suggest an unprotonated state<ref name="8a"/>. Furthermore, Glu297 forms a hydrogen bond with a water molecule within the active site as well as with Lys205, which is in turn hydrogen bonded to C1 carboxylate group of chorismate and the oxygen of the nucleophilic water molecule<ref name="6a"/><ref name="8a"/>. The glutamic residue (Gly252) could protonate the C4 leaving hydroxyl group. The pKa of Gly252 (7.7) suggest that is it is the only protonated glutamate residue in the active site at pH 7 and thus able to protonate the C4 leaving group<ref name="8a"/>. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5<ref name="8a"/>.

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'''Chorismate mutase (CM)'''

A magnesium ion in the active site orients the C1 carboxyl group of chorismate (Figure 8). A lysine residue then serves as a general base for the activation of a water molecule to attack at C2. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products.

A magnesium ion in the active site orients the C1 carboxyl group of chorismate (Figure 8)<ref name="6a"/><ref name="8a"/>. A lysine residue then serves as a general base for the activation of a water molecule to attack at C2<ref name="6a"/><ref name="8a"/>. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent<ref name="8a"/>. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion<ref name="6a"/><ref name="8a"/><ref name="2a"/>. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity<ref name="6a"/><ref name="8a"/><ref name="2a"/>. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products<ref name="1a"/><ref name="3a"/><ref name="5a"/>.

[[Image:CM2.png|450 px|center|thumb|'''Figure 8''': Isochorismate synthase activity <ref name="8a"/>.]]

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@@ Line 43: / Line 43: @@
 '''Isochorismate synthase (IS)'''
-Currently, isochorismate is believed to be formed from chorismate through a proposed Sn2 mechanism involving nucleophilic attack of an activated water molecule to the C2 center followed by either a concerted or stepwise elimination of the C4 hydroxyl group (Figure 7)<ref name="9a"/><ref name="6a"/><ref name="8a"/>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons<ref name="6a"/><ref name="8a"/>. However, mutational analysis of Lys205 suggested that the lysine reside is not the sole determinant in the activation of a water molecule for nucleophilic attack of the C2 center. Studies have shown that Lys205 is protonated at neutral pH and therefore can't act as a base to activate the water molecule, agreeing with the mutational analysis data. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule. The magnesium ion forces the negatively charged Glu297 residue to face toward the active site and the pKa of Glu297 (3.9) suggest an unprotonated state. Furthermore, Glu297 forms a hydrogen bond with a water molecule within the active site as well as with Lys205, which is in turn hydrogen bonded to C1 carboxylate group of chorismate and the oxygen of the nucleophilic water molecule. The glutamic residue (Gly252) could protonate the C4 leaving hydroxyl group. The pKa of Gly252 (7.7) suggest that is it is the only protonated glutamate residue in the active site at pH 7 and thus able to protonate the C4 leaving group. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5.
+Currently, isochorismate is believed to be formed from chorismate through a proposed Sn2 mechanism involving nucleophilic attack of an activated water molecule to the C2 center followed by either a concerted or stepwise elimination of the C4 hydroxyl group (Figure 7)<ref name="9a"/><ref name="6a"/><ref name="8a"/>. Lys205 has been proposed to act as the catalytic base, activating a water molecule in the active site by abstracting one of its protons<ref name="6a"/><ref name="8a"/>. However, mutational analysis of Lys205 suggested that the lysine reside is not the sole determinant in the activation of a water molecule for nucleophilic attack of the C2 center<ref name="6a"/><ref name="8a"/>. Studies have shown that Lys205 is protonated at neutral pH and therefore can't act as a base to activate the water molecule, agreeing with the mutational analysis data<ref name="1a"/><ref name="2a"/>. Instead of Lys205, Glu297 residue has been proposed to act as a base in the activation of the water molecule<ref name="6a"/><ref name="8a"/>. The magnesium ion forces the negatively charged Glu297 residue to face toward the active site and the pKa of Glu297 (3.9) suggest an unprotonated state<ref name="8a"/>. Furthermore, Glu297 forms a hydrogen bond with a water molecule within the active site as well as with Lys205, which is in turn hydrogen bonded to C1 carboxylate group of chorismate and the oxygen of the nucleophilic water molecule<ref name="6a"/><ref name="8a"/>. The glutamic residue (Gly252) could protonate the C4 leaving hydroxyl group. The pKa of Gly252 (7.7) suggest that is it is the only protonated glutamate residue in the active site at pH 7 and thus able to protonate the C4 leaving group<ref name="8a"/>. The pKa of Gly252 also accounts for the accumulation of isochorismate at pH values below 7.5<ref name="8a"/>.
@@ Line 50: / Line 50: @@
 '''Chorismate mutase (CM)'''
-A magnesium ion in the active site orients the C1 carboxyl group of chorismate (Figure 8). A lysine residue then serves as a general base for the activation of a water molecule to attack at C2. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products.
+A magnesium ion in the active site orients the C1 carboxyl group of chorismate (Figure 8)<ref name="6a"/><ref name="8a"/>. A lysine residue then serves as a general base for the activation of a water molecule to attack at C2<ref name="6a"/><ref name="8a"/>. The catalytic mechanism for conversion of isochorismate to salicylate by MbtI is a sigmatropic, pericyclic mechanism that is pH-dependent<ref name="8a"/>. Chromate mutase activity is only observed in the absence of magnesium ion in the active site while salicylate synthase activity is depended on magnesium ion<ref name="6a"/><ref name="8a"/><ref name="2a"/>. The active site of MbtI is altered by the removal of the magnesium cofactor causing chromate mutase activity<ref name="6a"/><ref name="8a"/><ref name="2a"/>. MbtI has differing binding modes for chromate that leads to different substrate conformations/transition states and resulting in different products<ref name="1a"/><ref name="3a"/><ref name="5a"/>.
 [[Image:CM2.png|450 px|center|thumb|'''Figure 8''': Isochorismate synthase activity <ref name="8a"/>.]]