1p48
From Proteopedia
OCA (Talk | contribs)
(New page: 200px<br /><applet load="1p48" size="450" color="white" frame="true" align="right" spinBox="true" caption="1p48, resolution 2.00Å" /> '''REVERSE PROTONATION ...)
Next diff →
Revision as of 21:20, 20 November 2007
|
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 MG and PEP 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
Page seeded by OCA on Tue Nov 20 23:27:48 2007
