Progress curves analysis as an alternative for exploration of activation-inhibition phenomena in cholinesterases.
The kinetic behaviour of insect acetylcholinesterases deviates from the Michaelis-Menten pattern. These deviations are known as activation or inhibition at various substrate concentrations and can be more or less observable depending on mutations around the active site of the enzyme. Most kinetic studies on these enzymes still rely on initial rate measurements. It is demonstrated here that according to this method one of the deviations can be overlooked. We attempt to point out that in such cases a detailed step-by-step progress curves analysis is successful. The study is focused on two different methods of analysing progress curves: (i) the first one is based on an integrated initial rate equation which can sufficiently fit truncated progress curves under corresponding conditions; and (ii) the other one precludes the algebraic formulae, but uses numerical integration for searching a non analytical solution of ordinary differential equations describing a kinetic model. All methods are tested on three different acetylcholinesterase mutants from Drosophila melanogaster. The results indicate that kinetic parameters for the E107K mutant with highly expressive activation and inhibition can be well evaluated applying any analysis method. It is quite different for E107W and E107Y mutants where latent activation is present, but discovered only using one or the other progress curves analysis methods.
Golicnik
M
M
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia. golicnik@ibmi.mf.uni-lj.si
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000122:Acetylthiocholine / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D003198:Computer Simulation; D004228:Dithionitrobenzoic Acid; D004331:Drosophila melanogaster / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D013439:Sulfhydryl Reagents
D013439:Sulfhydryl Reagents; D000122:Acetylthiocholine; D004228:Dithionitrobenzoic Acid; D000110:Acetylcholinesterase