KINETIC MODELS ON ACETYLCHOLINESTERASE MODULATION BY SELF-SUBSTRATE AND POLYAMINES: ESTIMATION OF INTERACTION PARAMETERS AND RATE CONSTANTS FOR FREE AND ACETYLATED STATES OF THE ENZYME

Abstract

Polyamines act as dual modulators on electric eel acetylcholinesterase, modifying both the apparent Km and Ki, depending on substrate levels. A kinetic model was developed to explain the results, based on two-step catalysis, a peripheral site for substrate inhibition apart from the catalytic site, and one binding site for polyamine. This model presented the best fittings to data, when compared with a simpler one considering one catalytic step. A fitting equation built up with sixteen independent parameters let us calculate the kinetic constants. In this way, we were able to solve the parameter identifiability problem arising from model uncertainty when only substrate was used in acetylcholinesterase kinetics. Besides, fitting parameters directly provide information about the binding constants of the different complexes, the modulatory strength of substrate and polyamines, and the effect on the standard activation free energy for acetylcholinesterase.Substrate inhibition operates mainly on the first catalytic step with an affinity constant of 5.2 mM-1, which is reduced to one third for the acetylated enzyme. The interaction factor between substrate binding at both sites is about 12. The modulatory strength of polyamines is spermine > spermidine > putrescine. This order is directly related to the number of amino groups in the molecule, and to the calculated free interaction energy. The effect of the number of amino groups on the binding energy is significantly increased in acetylated acetylcholinesterase. It is also inferred that the formation of a quaternary complex enzyme-substrate-substrate-polyamine would not be possible. Some relations between polyamine structure and acetylcholinesterase activity are suggested from estimated constants. Due to the distal amino group distances, it is possible for spermine and spermidine to span along the catalytic gorge of acetylcholinesterase, binding to the catalytic and peripheral sites in a way similar to bisquaternary ammonium inhibitors.