Expression of recombinant acetylcholinesterase in a baculovirus system: kinetic properties of glutamate 199 mutants

Abstract

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.

Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center

“…On the other hand, replacement of E202 by glutamine resulted in a marked decrease of the phosphylation rates. Radic et al have recently reported similar effects for the reactivity of an analogous mutant of TcAChE (E199Q) with DFP [29]. Again, as for the acylation process, we find that the effect of replacement of E450 on the rates of phosphylation (Table II), parallels that of E202 substitution.…”

“…As shown previously [18], replacement of the aromatic residue Y337 had only a marginal effect on the kinetic parameters (K m and k~at) for the two substrates (Table I) while the corresponding values for hydrolysis of ATC catalyzed by E202Q HuAChE [16], or of the corresponding mutation E199Q in TcAChE [29], differ significantly from those of the wild type enzyme. We now demonstrate that this decrease in catalytic efficiency of E202Q is observed also for the noncharged substrate TB (Table I), This indicates that the effect of replacement of E202 of HuAChE on catalysis is not solely due to electrostatic interactions with the substrate.…”

“…These residues include the catalytic triad ($203, H447 and E334), the putative oxyanion hole (GI21, G122 and A204), the pockets accommodating the alkoxy groups (W86, Y337 and F338), and residues which constitute the acyl pocket for ACh (F295 and F297) as well as E202 which is proximal to the P-O-C linkage. The participation of most of these residues in the analogous process of acylation was recently demonstrated experimentally, by site directed mutagenesis [15][16][17]29,35]. The kinetic studies reported here suggest that in phosphylation and even in the dealkylation of the OP conjugates of HuAChE, residue Y337 appears to have a minimal contribution.…”

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

“…Thus, these results are consistent with the structure and interaction energy calculations, which suggested that Glu202 stabilizes the O-isopropyl moiety of the sarin adduct in a conformation that enables nucleophilic attack on the phosphorus atom by HI-6. Glu202 has previously been reported as a key residue of AChE with an important role in catalysis (21), phosphylation (22), and aging (23). Here, we also observed, in addition to the interaction with the O-isopropyl moiety, an interaction between Glu202 and the methylene of Ser203 with an interaction energy of ∼7 kcal/mol (Table S4).…”

Design of acetylcholinesterases for biosensor applications

“…AChEs from Torpedo californica (Radic et al, 1992), Drosophila melanogaster (Chaabihi et al, 1994;Estrada-Mondaca et al, 1998) and rat brain could be actively expressed in satisfactory amounts in a baculovirus-insect cell system ( Table 2). The baculovirus system has severe limitations with respect to the production of larger amounts of protein, but seems to be very suitable for producing mutant recombinant AChEs.…”