1p48
From Proteopedia
(New page: 200px<br /><applet load="1p48" size="450" color="white" frame="true" align="right" spinBox="true" caption="1p48, resolution 2.00Å" /> '''REVERSE PROTONATION ...) |
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| - | [[Image:1p48.gif|left|200px]]<br /><applet load="1p48" size=" | + | [[Image:1p48.gif|left|200px]]<br /><applet load="1p48" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1p48, resolution 2.00Å" /> | caption="1p48, resolution 2.00Å" /> | ||
'''REVERSE PROTONATION IS THE KEY TO GENERAL ACID-BASE CATALYSIS IN ENOLASE'''<br /> | '''REVERSE PROTONATION IS THE KEY TO GENERAL ACID-BASE CATALYSIS IN ENOLASE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The pH dependence of enolase catalysis was studied to understand how | + | The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction. |
==About this Structure== | ==About this Structure== | ||
| - | 1P48 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with MG and PEP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Phosphopyruvate_hydratase Phosphopyruvate hydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.11 4.2.1.11] Full crystallographic information is available from [http:// | + | 1P48 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=PEP:'>PEP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Phosphopyruvate_hydratase Phosphopyruvate hydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.11 4.2.1.11] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1P48 OCA]. |
==Reference== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Saccharomyces cerevisiae]] | [[Category: Saccharomyces cerevisiae]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Cleland, W | + | [[Category: Cleland, W W.]] |
| - | [[Category: Larsen, T | + | [[Category: Larsen, T M.]] |
| - | [[Category: Poyner, R | + | [[Category: Poyner, R R.]] |
| - | [[Category: Reed, G | + | [[Category: Reed, G H.]] |
| - | [[Category: Sims, P | + | [[Category: Sims, P A.]] |
[[Category: MG]] | [[Category: MG]] | ||
[[Category: PEP]] | [[Category: PEP]] | ||
[[Category: beta barrel]] | [[Category: beta barrel]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:24:54 2008'' |
Revision as of 12:24, 21 February 2008
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REVERSE PROTONATION IS THE KEY TO GENERAL ACID-BASE CATALYSIS IN ENOLASE
Overview
The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.
About this Structure
1P48 is a Single protein structure of sequence from Saccharomyces cerevisiae with and as ligands. Active as Phosphopyruvate hydratase, with EC number 4.2.1.11 Full crystallographic information is available from OCA.
Reference
Reverse protonation is the key to general acid-base catalysis in enolase., Sims PA, Larsen TM, Poyner RR, Cleland WW, Reed GH, Biochemistry. 2003 Jul 15;42(27):8298-306. PMID:12846578
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