PDBsum entry: 1p43

Lyase

PDB id: 1p43

Contents

Protein chains

436 a.a. *

Ligands

2PG ×2

Metals

_MG ×4

Waters ×867

* Residue conservation analysis

PDB id:

1p43

Name:

Lyase

Title:

Reverse protonation is the key to general acid-base catalysi enolase

Structure:

Enolase 1. Chain: a, b. Synonym: 2-phosphoglycerate dehydratase, 2-phospho-d- glyce hydro-lyase. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: eno1 or enoa or hsp48 or ygr254w or g9160. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.185 R-free: 0.213

Authors:

P.A.Sims,T.M.Larsen,R.R.Poyner,W.W.Cleland,G.H.Reed

Key ref:

P.A.Sims et al. (2003). Reverse protonation is the key to general acid-base catalysis in enolase. Biochemistry, 42, 8298-8306. PubMed id: 12846578 DOI: 10.1021/bi0346345

Date:

21-Apr-03 Release date: 18-Nov-03

Protein chains

P00924  (ENO1_YEAST) -  Enolase 1

Seq: 437 a.a.

Struc: 436 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.4.2.1.11 - Phosphopyruvate hydratase.
• Reaction: 2-phospho-D-glycerate = phosphoenolpyruvate + H₂O

2-phospho-D-glycerate (Bound ligand (Het Group name = 2PG) corresponds exactly) = phosphoenolpyruvate + H(2)O

• Cofactor: Magnesium

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (5 terms)
• Biological process: regulation of vacuole fusion, non-autophagic (3 terms)
• Biochemical function: protein binding (5 terms)

Added reference

DOI no: 10.1021/bi0346345

Biochemistry 42:8298-8306 (2003)

PubMed id: 12846578

Reverse protonation is the key to general acid-base catalysis in enolase.

P.A.Sims, T.M.Larsen, R.R.Poyner, W.W.Cleland, G.H.Reed.

ABSTRACT

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.