1elx
From Proteopedia
(New page: 200px<br /><applet load="1elx" size="450" color="white" frame="true" align="right" spinBox="true" caption="1elx, resolution 2.6Å" /> '''E. COLI ALKALINE PHOS...) |
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| - | [[Image:1elx.gif|left|200px]]<br /><applet load="1elx" size=" | + | [[Image:1elx.gif|left|200px]]<br /><applet load="1elx" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1elx, resolution 2.6Å" /> | caption="1elx, resolution 2.6Å" /> | ||
'''E. COLI ALKALINE PHOSPHATASE MUTANT (S102A)'''<br /> | '''E. COLI ALKALINE PHOSPHATASE MUTANT (S102A)'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific | + | Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile. |
==About this Structure== | ==About this Structure== | ||
| - | 1ELX 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 ZN, MG and PO4 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http:// | + | 1ELX 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=ZN:'>ZN</scene>, <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=PO4:'>PO4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1ELX OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Brennan, C.]] | [[Category: Brennan, C.]] | ||
[[Category: Hehir, M.]] | [[Category: Hehir, M.]] | ||
| - | [[Category: Kantrowitz, E | + | [[Category: Kantrowitz, E R.]] |
[[Category: Nolte, M.]] | [[Category: Nolte, M.]] | ||
[[Category: Stec, B.]] | [[Category: Stec, B.]] | ||
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[[Category: hydrolase]] | [[Category: hydrolase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:29:10 2008'' |
Revision as of 10:29, 21 February 2008
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E. COLI ALKALINE PHOSPHATASE MUTANT (S102A)
Overview
Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.
About this Structure
1ELX is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Alkaline phosphatase, with EC number 3.1.3.1 Full crystallographic information is available from OCA.
Reference
Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102., Stec B, Hehir MJ, Brennan C, Nolte M, Kantrowitz ER, J Mol Biol. 1998 Apr 3;277(3):647-62. PMID:9533886
Page seeded by OCA on Thu Feb 21 12:29:10 2008
Categories: Alkaline phosphatase | Escherichia coli | Single protein | Brennan, C. | Hehir, M. | Kantrowitz, E R. | Nolte, M. | Stec, B. | MG | PO4 | ZN | Hydrolase
