Kinetic Mechanism And Application On Acetylcholinesterase

Determination of Acetylcholinesterase's activity by Ellman's method to study the kinectic mechanism of Acetylcholinesterase, several parameters are analysed, such as Michaelis constant Km, Maximal Velocity Vmax, Turnover number Kcat, Enzymic specific constant Kcat/Km, Biomolecular rate constant Ki,, Affinity constant of the inhibitior for the active site of Acetylcholinesterase Ka, Phosphorylation or carbamylation rate constant K2. Based on these kinetic constants, the resistant mechanism to organophosphates or carbamates is digging up. The alteration of Enzymic specific constant is the test standard for the change of Acetylcholinesterase's catalytic efficiency to establish model for selecting effective insecticides. Based on the kinetic model, comparison of Acetylcholinesterase from susceptible housefly strain, resistant housefly strain, California Torpedo different insensitivity towards 16 kinds of organophosphates or carbamates, the chemical structure bearing lower toxicity to vertebrate but higher toxicity to harmful insects are studied. The major results are summarized as following:1,According to the spectrum of resistance, high-level cross-resistant to OPs was encountered in Musca domestica selected for resistance to CBs which suggests the resistance was caused by decreased sensibility of AChE.2,The study on AChE from resistant strain exhibits temperature optimum at 37°C, while susceptible strain exhibits temperature optimum at 34°C; difference was also investigated for the hydrolysis of AChE-catalyzed from resistant and susceptible strain.3,The research on the kinetic analysis suggests that AChE from resistant strain might be qualitatively altered, resulting in decreased affinity and sensitivity to OPs and CBs, which is the experimental proof of the resistance.4,Application of Ki to study the structure-activity relationships of OPs and CBs as inhibitor.5,Based on the character and kinetic parameters of AChE from resistant and susceptible strain, vertebrate (Torpedo), selection model was established to screen anti-resistant and highly selective compounds, which make the screening highly effective.6,The results show that decreased sensitivity of Acetylcholinesterase is associated with decreased catalytic efficiency of enzyme from resistant strain, mainly caused by the decreased affinity to enzyme. The studies show that Dichlorvos and Chlorpyrifos-oxon has the character of higher toxicity to resistant insects but lower toxicity to Torpedo, which suggests a new theoretic way for designing and selecting of green pesticide.