PMID- 18775727
OWN - NLM
STAT- MEDLINE
DCOM- 20081106
LR  - 20161124
IS  - 1089-8638 (Electronic)
IS  - 0022-2836 (Linking)
VI  - 383
IP  - 3
DP  - 2008 Nov 14
TI  - Insights into the catalytic mechanism of human sEH phosphatase by site-directed 
      mutagenesis and LC-MS/MS analysis.
PG  - 627-40
LID - 10.1016/j.jmb.2008.08.049 [doi]
AB  - We have recently reported that human soluble epoxide hydrolase (sEH) is a 
      bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a 
      structural relationship with other members of the haloacid dehalogenase 
      superfamily, the sEH N-terminal phosphatase domain revealed four conserved 
      sequence motifs, including the proposed catalytic nucleophile D9, and several 
      other residues potentially implicated in substrate turnover and/or Mg(2+) 
      binding. To enlighten the catalytic mechanism of dephosphorylation, we 
      constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A 
      total of 18 mutants were constructed and recombinantly expressed in Escherichia 
      coli as soluble proteins, purified to homogeneity and subsequently analysed for 
      their kinetic parameters. A replacement of residues D9, K160, D184 or N189 
      resulted in a complete loss of phosphatase activity, consistent with an essential 
      function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 
      leads to sEH mutant proteins with altered kinetic properties. We further provide 
      evidence of the formation of an acylphosphate intermediate on D9 by liquid 
      chromatography-tandem mass spectrometry based on the detection of homoserine 
      after NaBH(4) reduction of the phosphorylated enzyme, which identifies D9 as the 
      catalytic nucleophile. Surprisingly, we could only show such homoserine formation 
      using the D11N mutant, which strongly suggests D11 to be involved in the 
      acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes 
      rate limiting, which then allows trapping of the labile intermediate. Substrate 
      turnover in the presence of (18)H(2)O revealed that the nucleophilic attack 
      during the second reaction step occurs at the acylphosphate phosphorous. Based on 
      these findings, we propose a two-step catalytic mechanism of dephosphorylation 
      that involves the phosphate substrate hydrolysis by nucleophilic attack by the 
      catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme 
      intermediate supported by D11.
FAU - Cronin, Annette
AU  - Cronin A
AD  - Institute of Pharmacology and Toxicology, University of Zurich, Zurich, 
      Switzerland. cronin@pharma.uzh.ch
FAU - Homburg, Shirli
AU  - Homburg S
FAU - Durk, Heike
AU  - Durk H
FAU - Richter, Ingrid
AU  - Richter I
FAU - Adamska, Magdalena
AU  - Adamska M
FAU - Frere, Frederic
AU  - Frere F
FAU - Arand, Michael
AU  - Arand M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20080827
PL  - Netherlands
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - 0 (Phosphates)
RN  - 0 (Protein Subunits)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.1.3.2 (Phosphoric Monoester Hydrolases)
RN  - EC 3.3.2.- (Epoxide Hydrolases)
SB  - IM
MH  - Amino Acid Sequence
MH  - Binding Sites
MH  - Chromatography, Liquid
MH  - Dimerization
MH  - Epoxide Hydrolases/*chemistry/genetics/*metabolism
MH  - Humans
MH  - Mass Spectrometry
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Phosphates/chemistry/metabolism
MH  - Phosphoric Monoester Hydrolases/chemistry/genetics/*metabolism
MH  - Protein Structure, Quaternary
MH  - *Protein Structure, Tertiary
MH  - Protein Subunits/chemistry/genetics/metabolism
MH  - Recombinant Proteins/chemistry/genetics/metabolism
MH  - Sequence Alignment
EDAT- 2008/09/09 09:00
MHDA- 2008/11/07 09:00
CRDT- 2008/09/09 09:00
PHST- 2008/06/16 00:00 [received]
PHST- 2008/08/13 00:00 [revised]
PHST- 2008/08/20 00:00 [accepted]
PHST- 2008/09/09 09:00 [pubmed]
PHST- 2008/11/07 09:00 [medline]
PHST- 2008/09/09 09:00 [entrez]
AID - S0022-2836(08)01059-0 [pii]
AID - 10.1016/j.jmb.2008.08.049 [doi]
PST - ppublish
SO  - J Mol Biol. 2008 Nov 14;383(3):627-40. doi: 10.1016/j.jmb.2008.08.049. Epub 2008 
      Aug 27.