engleski

Kinetic analysis of the reaction of butyrylcholinesterase with specific substrates

Butyrylcholinesterase (BChE; EC 3.1.1.8) catalyses the hydrolysis of its two specific substrates, butyrylthiocholine (BTCh) and benzoylcholine (BzCh); the hydrolysis rate of the first being a sigmoidal function of substrate concentration and that of the latter bell-shaped. The activity of human serum usual and atypical BChE and horse serum BChE was measured at 0.01 to 100 mM butyrylthiocholine and benzoylcholine. In order to calculate catalytic constants four equations were applied: eqn. 1 describes the relationship between enzyme activity and substrate concentration for a simple mechanism where there is binding of one substrate molecule to a single catalytic site; eqn. 2 assumes enzyme inhibition by the excess of substrate concentration; Hill eqn. assumes co-operativity between the substrate binding sites of the enzyme and eqn. 4 relies on the assumption of the binding of an additional substrate molecule to the acylated enzyme intermediate or to a peripheral regulatory site. The binding of a second substrate molecule causes either an activation or inhibition of the enzyme/substrate reaction. Data were fitted by a non-linear regression to the eqns. 1-4 and the catalytic constants were calculated. For the total range of substrate concentrations data obtained with BTCh poorly fitted eqn. l, but fitted well both eqns. 3 and 4. Data obtained with BzCh poorly fitted eqn.l (0.01-10 mM BzCh) and eqn. 2 (10-100 mM BzCh). The results support the premise of two binding sites on BChE.

butyrylcholinesterase; PS-curves; benzoylcholine; activation; inhibition; catalytic constants

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