User:Adam Mirando/Sandbox 1: Difference between revisions

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== Mechanism ==

===Substrate Binding and Intermediate Stabilization===

Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several hydrophobic residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.

The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the alloxanthine inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in Km and a 92.6% reduction in kcat compared to the wild type enzyme <ref name="hypoxanthine" />. The hypoxanthine oxidation mechanism, however, shows an opposite response to the mutations. The E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in Km and a 98.9% reduction in kcat). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.

===Xanthine Oxidation Mechanism===

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The oxidation of hypoxanthine is theorized to occur much like the oxidation of xanthine. Once again the mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1262. The deprotonated molybdenum oxygen is then free to engage in a nucleophilic attack on C2 of hypoxanthine paired with the concomitant hydride transfer from hypoxanthine to the molybdenum bound sulfur. The reactive species are stabilized by hydrogen bond interactions between the protonated Glu1262 and N2 of hypoxanthine as well as between Glu803 and N5 and the carbonyl of C4 of hypoxanthine. The subsequent electron transfer and product release occur similarly to the xanthine oxidation reaction <ref name="hypoxanthine">PMID:17301077</ref>.

~~===Xanthine Binding and Intermediate Stabilization===~~

~~<applet load='3BDJ' size='300' frame='true' align='right' caption='Insert caption here' />~~

Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several hydrophobic residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.

The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the alloxanthine inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in Km and a 92.6% reduction in kcat compared to the wild type enzyme <ref name="hypoxanthine" />. In the case of the hypoxanthine oxidation mechanism, however, E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in Km and a 98.9% reduction in kcat). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.

===Redox Potential===

@@ Line 22: / Line 22: @@
 == Mechanism ==
+===Substrate Binding and Intermediate Stabilization===
+<applet load='3BDJ' size='300' frame='true' align='right' caption='Insert caption here' />
+Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several hydrophobic residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.
+	The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the alloxanthine inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in K<sub>m</sub> and a 92.6% reduction in k<sub>cat</sub> compared to the wild type enzyme <ref name="hypoxanthine" />.  The hypoxanthine oxidation mechanism, however, shows an opposite response to the mutations. The E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in K<sub>m</sub> and a 98.9% reduction in k<sub>cat</sub>). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.
 ===Xanthine Oxidation Mechanism===
@@ Line 34: / Line 39: @@
 The oxidation of hypoxanthine is theorized to occur much like the oxidation of xanthine. Once again the mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1262. The deprotonated molybdenum oxygen is then free to engage in a nucleophilic attack on C2 of hypoxanthine paired with the concomitant hydride transfer from hypoxanthine to the molybdenum bound sulfur. The reactive species are stabilized by hydrogen bond interactions between the protonated Glu1262 and N2 of hypoxanthine as well as between Glu803 and N5 and the carbonyl of C4 of hypoxanthine. The subsequent electron transfer and product release occur similarly to the xanthine oxidation reaction <ref name="hypoxanthine">PMID:17301077</ref>.
-===Xanthine Binding and Intermediate Stabilization===
-<applet load='3BDJ' size='300' frame='true' align='right' caption='Insert caption here' />
-Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several hydrophobic residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.
-	The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the alloxanthine inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in K<sub>m</sub> and a 92.6% reduction in k<sub>cat</sub> compared to the wild type enzyme <ref name="hypoxanthine" />.  In the case of the hypoxanthine oxidation mechanism, however, E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in K<sub>m</sub> and a 98.9% reduction in k<sub>cat</sub>). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.
 ===Redox Potential===