engleski

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

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH=7.4 at 25 oC. 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 obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (1), Hill equation (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 a range from 0.01 to 50 mM BTCh, the activity vs. substrate concentration relationship deviated from Michaelis-Menten kinetics (Eqn.1) while data fitted well with Eqns. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges; the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01 - 1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0 - 50 mM BTCh). Hill coefficients (nH) calculated from Eqn. 2 were similar for all phenotypes (nH @ 0.5). The dissociation 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 ) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

butyrylcholinesterase; human serum phenotypes/genotypes; catalytic constants; substrat activation/inhibition

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