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 degrees C. 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 (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 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 Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 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.
Simeon-Rudolf
V
V
Institute for Medical Research and Occupational Health, Zagreb, Croatia. vesimeon@mimi.imi.hr
Reiner
E
E
Evans
R T
RT
George
P M
PM
Potter
H C
HC
D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000097:blood* / Q000235:genetics; D002092:Butyrylthiocholine / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016014:Linear Models; D008956:Models, Chemical; D010641:Phenotype; D013379:Substrate Specificity
D002800:Cholinesterase Inhibitors; D002092:Butyrylthiocholine; D002091:Butyrylcholinesterase