Expression of recombinant acetylcholinesterase in a baculovirus system: kinetic properties of glutamate 199 mutants.
The glycophospholipid-linked, amphiphilic form of acetylcholinesterase (AChE) from Torpedo californica and the hydrophilic form from mouse were overexpressed in Sf9 insect cells using the baculovirus expression system. Recombinant baculovirus, constructed by inserting AChE cDNA's into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedron promoter, yielded recombinant enzyme varying between 0.5 and 3.8 mg/L. The recombinant enzyme was glycosylated although it migrated slightly more rapidly in SDS gel electrophoresis than enzyme purified from the electric organ of Torpedo. Kinetic properties of the recombinant DNA- and tissue-derived enzymes are identical. The detailed catalytic properties and susceptibility to inhibitors were examined for two enzyme mutations of the glutamate residue N-terminal to the active site serine. The Glu199 to Gln mutation shifted both the Km and Kss to higher substrate concentrations and resulted in a kcat of 28% of the wild type. Mutation of Glu199 to Asp also yielded a reduction in kcat but with no change in Km. Substrate inhibition normally apparent in wild-type AChE was eliminated with the Asp mutation, suggesting that substrate catalysis and substrate inhibition are not directly linked. Both mutations decreased the affinity of reversible inhibitors and reduced the rates of phosphorylation and carbamoylation; these changes were more striking with the Gln199 mutation. Decarbamoylation rates were unaffected by these mutations. Glu199 is the charged residue found deep within the active center gorge close to the site of acetylcholine binding, and our findings indicate it influences, but is not essential for, efficient catalysis.
Radić
Z
Z
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Gibney
G
G
Kawamoto
S
S
MacPhee-Quigley
K
K
Bongiorno
C
C
Taylor
P
P
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006031:Glycosylation; D007700:Kinetics; D051379:Mice; D009036:Moths; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014101:Torpedo; D014961:X-Ray Diffraction
D002800:Cholinesterase Inhibitors; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000110:Acetylcholinesterase