title: Conformational rigidity of cholinesterases allows for the prediction of combined effects in a particular double mutant.
abstract: The conformational rigidity of Drosophila melanogaster AChE, was checked by kinetic means on recombinant enzyme with the substitutions of two important amino acids, one at the catalytic anionic site (W83A), one at the peripheral anionic site (W321A) and the double mutant with both tryptophans substituted by alanines (W83A/W321A). It was hypothesized that the individual mutations would affect only the binding affinities of substrate molecules at each site and that a predictable effect would show up in the corresponding double mutant. Simple inspection revealed that bell shaped curves of activity at wide substrate concentration range in the catalytic anionic site mutants carry much less information than the analogous asymmetric ones of the wild type and peripheral anionic site mutant. Therefore, a concurrent kinetic analysis of the curves for all four enzymes was undertaken by constraining mutation independent parameters: unchanged affinity at the catalytic/peripheral anionic site of the opposite mutant in comparison to the parameters for wild type enzyme. Additionally, the parameters for W83A mutated enzyme were employed for the characterization of double mutant (W83A/W321A) protein by setting the dissociation constant for the substrate at the peripheral anionic site as determined for W321A mutant. Simultaneous analysis exactly reproduced the behavior of the double mutant without any significant change of previously reported values for the wild type enzyme (Stojan et al., 2004). This kinetic behavior is completely in line with the crystallographic evidence of structural rigidity in cholinesterases.
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, University of Ljublajna, Vrazov trg 2, Ljubljana, Slovenia. Electronic address: stojan@mf.uni-lj.si.
authors.forename: Jure
authors.initials: J
authors.lastname: Stojan
mesh_terms: D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D002802:Cholinesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D002802:Cholinesterases
keywords: Drosophila melanogaster acetylcholinesterase; Enzyme kinetics; Reaction mechanism; Site directed mutations