Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability.
The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
Centre de Recherches du Service de Santé des Armées, Unité d'enzymologie, France. cclerybarraud@crssa.net
Cécile
C
Cléry-Barraud
Arie
A
Ordentlich
Haim
H
Grosfeld
Avigdor
A
Shafferman
Patrick
P
Masson
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D004795:Enzyme Stability; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000378:metabolism; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C031762:1-naphthylamine-8-sulfonic acid; D000110:Acetylcholinesterase