1cqi: Difference between revisions

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CRYSTAL STRUCTURE OF THE COMPLEX OF ADP AND MG2+ WITH DEPHOSPHORYLATED E. COLI SUCCINYL-COA SYNTHETASE

OverviewOverview

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA + P(i) + NDP <--> succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (>15 x K(d)) was added; Mg(2+) ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+) ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+) complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound in the ATP-grasp fold of the N-terminal domain of each beta-subunit (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We describe the specific interactions of the nucleotide-Mg(2+) complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the "down" position where it can interact with the nucleotide approximately 35 A from where His 246alpha is seen in both phosphorylated and dephosphorylated SCS.

About this StructureAbout this Structure

1CQI is a Protein complex structure of sequences from Escherichia coli with , , and as ligands. Active as Succinate--CoA ligase (ADP-forming), with EC number 6.2.1.5 Full crystallographic information is available from OCA.

ReferenceReference

ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by x-ray crystallography., Joyce MA, Fraser ME, James MN, Bridger WA, Wolodko WT, Biochemistry. 2000 Jan 11;39(1):17-25. PMID:10625475

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-[[Image:1cqi.gif|left|200px]]<br /><applet load="1cqi" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1cqi.gif|left|200px]]<br /><applet load="1cqi" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1cqi, resolution 3.30&Aring;" />
 '''CRYSTAL STRUCTURE OF THE COMPLEX OF ADP AND MG2+ WITH DEPHOSPHORYLATED E. COLI SUCCINYL-COA SYNTHETASE'''<br />
 ==Overview==
-Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction, via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA +, P(i) + NDP &lt;--&gt; succinate + CoA + NTP (N denotes adenosine or guanosine)., To determine the structure of the enzyme with nucleotide bound, crystals, of phosphorylated Escherichia coli SCS were soaked in successive, experiments adopting progressive strategies. In the first experiment, 1 mM, ADP (&gt;15 x K(d)) was added; Mg(2+) ions were omitted to preclude the, formation of an insoluble precipitate with the phosphate and ammonium, ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+), ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+), complex. In the successful experiment, the phosphate buffer was exchanged, with MOPS, the concentration of sulfate ions was lowered, and the, concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound, in the ATP-grasp fold of the N-terminal domain of each beta-subunit, (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We, describe the specific interactions of the nucleotide-Mg(2+) complex with, SCS, compare these results with those for other proteins containing the, ATP-grasp fold, and present a hypothetical model of the, histidine-containing loop in the "down" position where it can interact, with the nucleotide approximately 35 A from where His 246alpha is seen in, both phosphorylated and dephosphorylated SCS.
+Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA + P(i) + NDP &lt;--&gt; succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (&gt;15 x K(d)) was added; Mg(2+) ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+) ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+) complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound in the ATP-grasp fold of the N-terminal domain of each beta-subunit (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We describe the specific interactions of the nucleotide-Mg(2+) complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the "down" position where it can interact with the nucleotide approximately 35 A from where His 246alpha is seen in both phosphorylated and dephosphorylated SCS.
 ==About this Structure==
-CQI is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MG, PO4, COA and ADP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate--CoA_ligase_(ADP-forming) Succinate--CoA ligase (ADP-forming)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=6.2.1.5 6.2.1.5] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1CQI OCA].
+CQI is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=PO4:'>PO4</scene>, <scene name='pdbligand=COA:'>COA</scene> and <scene name='pdbligand=ADP:'>ADP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate--CoA_ligase_(ADP-forming) Succinate--CoA ligase (ADP-forming)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=6.2.1.5 6.2.1.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CQI OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Protein complex]]
 [[Category: Succinate--CoA ligase (ADP-forming)]]
-[[Category: Bridger, W.A.]]
+[[Category: Bridger, W A.]]
-[[Category: Fraser, M.E.]]
+[[Category: Fraser, M E.]]
-[[Category: James, M.N.G.]]
+[[Category: James, M N.G.]]
-[[Category: Joyce, M.A.]]
+[[Category: Joyce, M A.]]
-[[Category: Wolodko, W.T.]]
+[[Category: Wolodko, W T.]]
 [[Category: ADP]]
 [[Category: COA]]
@@ Line 27: / Line 27: @@
 [[Category: rossmann fold]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 12:40:41 2007''
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