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Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase phenotypes

The hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) phenotypes was measured in phosphate buffer pH=7.4. Three sera were used for each phenotype taken from individuals whose cholinesterase phenotype had previously been determined by measurement of the activities and inhibition by dibucain, sodium fluoride and the dimethylcarbamate Ro 02-0683. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To calculated the catalytic constants three equations were applied: Michaelis-Menten (1), Hill (2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (3). Over the range from 0.01 to 100 mM BTCh the activity vs. substrate concentration relationship deviated from MichaelisMenten kinetics while the pS-curves fitted well with eqns. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges. The Km constants for the five phenotypes ranged from 0.1 to 0.2 mM (at 0.01 - 1.0 mM BTCh) and from 0.4 to 2.0 mM (at 1.0 - 50 mM BTCh). Hill coefficients calculated from eqn.2 were similar for all phenotypes. The binding constants K1 and K2 calculated from eqn. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1 ) and 0.89 and 4.9 mM (K2 ). The Vm values calculated from all three equations agreed with maximal activities obtained experimentally. Application of the three equations to our experimental data supports the argument that butyrylcholinesterase has two substrate binding sites.

butyrylcholinesterase; human serum cholinesterase phenotypes/genotypes; substrate binding sites; catalytic constants

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