1tpc: Difference between revisions
New page: left|200px<br /><applet load="1tpc" size="450" color="white" frame="true" align="right" spinBox="true" caption="1tpc, resolution 1.9Å" /> '''OFFSET OF A CATALYTIC... |
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[[Image:1tpc.jpg|left|200px]]<br /><applet load="1tpc" size=" | [[Image:1tpc.jpg|left|200px]]<br /><applet load="1tpc" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1tpc, resolution 1.9Å" /> | caption="1tpc, resolution 1.9Å" /> | ||
'''OFFSET OF A CATALYTIC LESION BY A BOUND WATER SOLUBLE'''<br /> | '''OFFSET OF A CATALYTIC LESION BY A BOUND WATER SOLUBLE'''<br /> | ||
==Overview== | ==Overview== | ||
The structural basis for the improvement in catalytic efficiency of the | The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules. | ||
==About this Structure== | ==About this Structure== | ||
1TPC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Gallus_gallus Gallus gallus] with PGH as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Triose-phosphate_isomerase Triose-phosphate isomerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.1.1 5.3.1.1] Full crystallographic information is available from [http:// | 1TPC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Gallus_gallus Gallus gallus] with <scene name='pdbligand=PGH:'>PGH</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Triose-phosphate_isomerase Triose-phosphate isomerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.1.1 5.3.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1TPC OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Triose-phosphate isomerase]] | [[Category: Triose-phosphate isomerase]] | ||
[[Category: Knowles, J | [[Category: Knowles, J R.]] | ||
[[Category: Komives, E | [[Category: Komives, E A.]] | ||
[[Category: Liu, K | [[Category: Liu, K D.]] | ||
[[Category: Petsko, G | [[Category: Petsko, G A.]] | ||
[[Category: Ringe, D.]] | [[Category: Ringe, D.]] | ||
[[Category: Sugio, S.]] | [[Category: Sugio, S.]] | ||
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[[Category: isomerase(intramolecular oxidoreductse)]] | [[Category: isomerase(intramolecular oxidoreductse)]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:15:58 2008'' | ||
Revision as of 16:15, 21 February 2008
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OFFSET OF A CATALYTIC LESION BY A BOUND WATER SOLUBLE
OverviewOverview
The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.
About this StructureAbout this Structure
1TPC is a Single protein structure of sequence from Gallus gallus with as ligand. Active as Triose-phosphate isomerase, with EC number 5.3.1.1 Full crystallographic information is available from OCA.
ReferenceReference
The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules., Komives EA, Lougheed JC, Liu K, Sugio S, Zhang Z, Petsko GA, Ringe D, Biochemistry. 1995 Oct 17;34(41):13612-21. PMID:7577950
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