1dhj
From Proteopedia
(New page: 200px<br /><applet load="1dhj" size="450" color="white" frame="true" align="right" spinBox="true" caption="1dhj, resolution 1.8Å" /> '''LONG-RANGE STRUCTURAL...) |
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| - | [[Image:1dhj.gif|left|200px]]<br /><applet load="1dhj" size=" | + | [[Image:1dhj.gif|left|200px]]<br /><applet load="1dhj" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1dhj, resolution 1.8Å" /> | caption="1dhj, resolution 1.8Å" /> | ||
'''LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE'''<br /> | '''LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | X-ray crystal structures have been determined for a second-site revertant | + | X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B. |
==About this Structure== | ==About this Structure== | ||
| - | 1DHJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CL, CA and MTX as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Dihydrofolate_reductase Dihydrofolate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.3 1.5.1.3] Full crystallographic information is available from [http:// | + | 1DHJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CL:'>CL</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=MTX:'>MTX</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Dihydrofolate_reductase Dihydrofolate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.3 1.5.1.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DHJ OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Brown, K | + | [[Category: Brown, K A.]] |
[[Category: Kraut, J.]] | [[Category: Kraut, J.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
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[[Category: oxidoreductase]] | [[Category: oxidoreductase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:16:39 2008'' |
Revision as of 10:16, 21 February 2008
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LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
Overview
X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.
About this Structure
1DHJ is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Dihydrofolate reductase, with EC number 1.5.1.3 Full crystallographic information is available from OCA.
Reference
Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase., Brown KA, Howell EE, Kraut J, Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11753-6. PMID:8265622
Page seeded by OCA on Thu Feb 21 12:16:39 2008
Categories: Dihydrofolate reductase | Escherichia coli | Single protein | Brown, K A. | Kraut, J. | CA | CL | MTX | Oxidoreductase
