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

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluori de-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinestera se (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degr ees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where the re 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 cat alytic site and to a peripheral site on the enzyme (Eq. 3). Over a range fr om 0.01 to 50 mM BTCh, the activity versus substrate concentration relation ship 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 K-m constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTC h) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (n(H)) cal culated from Eq. 2 were similar for all phenotypes (n(H) congruent to 0.5). The dissociation constants K-1 and K-2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K-1) and 0.89 and 4.9 mM (K-2 ) for the five phenotypes. Experimental data support the assumption that th e phenotypes studied have two substrate binding sites. (C) 1999 Elsevier Sc ience Ireland Ltd.; All rights reserved.