Mechanism of eserine action on the hydrolysis of butyrylthiocholine by butyrylcholinesterase.

The mechanism of the interaction of eserine with butyrylcholinesterase has been proposed only on the basis of analogy with acetylcholinesterase. Here the interactions was studied in detail and the results analysed by classical kinetic methods and by means of mathematical modelling. An appropriate kinetic scheme was developed, an adequate equation derived and the corresponding kinetic parameters evaluated. The findings suggest that a fast but relatively weak binding of eserine to the enzyme's active site is followed by a slow acylation step and by an even slower rate limiting deacylation step so misrepresenting eserine as an irreversible inhibitor. The proposed kinetic scheme also suggests that the reaction of eserine with a peripheral substrate site is unlikely as seen with the substrate, butyrylthiocholine.

The influence of an organosulfonate on the reaction between Ellman's reagent and thiocholine.

The aim of this work was to elucidate the effect of methanesulfonyl fluoride on the detection reaction for the determination of cholinesterase activity. The effect of methanesulfonyl fluoride was studied by monitoring the time course of the appearance and disappearance of the detection reaction product using a stopped-flow technique. The obtained experimental data were analyzed by progress curve analysis. It was found that the effect of methanesulfonyl fluoride results from a reaction between methanesulfonyl fluoride and the detection reaction product with the following rate constant: k1 = 5.33 x 10(-2) M-1s-1. In evaluating the effect of methanesulfonyl fluoride, the decomposition of this agent in water was included in the analysis because the decomposition considerably affected the reaction between methanesulfonyl fluoride and the product. The corresponding rate constant was found to be k2 = 4.9 x 10(-5) M-1s-1.

Velocity of Ellman's reaction and its implication for kinetic studies in the millisecond time range.

A detailed study of the velocity of the reaction between Ellman's reagent and thiocholine was undertaken, in order to test the possibilities of this reaction as a detection method for the earlier stages of cholinesterases reactions. Experiments were carried out on a stopped-flow apparatus with a built-in spectrophotometer. The obtained experimental data were analyzed by fitting the data to theoretical kinetic equations derived for the reaction. In this way, a complete kinetic characterization of the reaction was obtained. An important practical result derived from our investigations is the finding that, under most experimental conditions, the Ellman's reaction is more than sufficiently rapid as a detection method. However, in the case of reactions in the time scale of 200 milliseconds or less, this being 5 times the half life of Ellman's reaction at standard conditions, one has to consider the interference of this reaction with the enzyme reaction itself.

On the inhibition of cholinesterase by D-tubocurarine.

Some investigation in this laboratory pointed to an unexpectedly slow inhibition of cholinesterase by D-tubocurarine, occurring in addition to a typically instantaneous inhibition. In order to elucidate this phenomenon, the hydrolysis of butyrylthiocholine catalyzed by cholinesterase was recorded, in the absence and presence of D-tubocurarine, on a stopped-flow apparatus. Experimental results were analyzed by classical kinetic methods and by means of mathematical modeling. It was found that the inhibition is of a double character, consisting of an instantaneous phase and a slow one occurring in a minute time scale. It seems that the action of D-tubocurarine is a consequence of an instantaneous binding of D-tubocurarine to a peripheral site, followed by a relatively slow conformational transition in the enzyme.

The role of hydration in an enzyme reaction.

The aim of the work was to elucidate the role of water in the reaction between acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and methanesulfonyl fluoride, accelerated by accelerators. The reaction between the enzyme and methanesulfonyl fluoride in the presence of individual monovalent cations of the Hofmeister series was investigated. The results obtained were analyzed in comparison with the effect of methanesulfonylation of the specific accelerators tetramethylammonium and tetraethylammonium under various experimental conditions. The monovalent cations of the Hofmeister series accelerate the reaction. Their effect--as well as that of specific accelerators--significantly correlates with the effect of these agents on the structure of water. These findings, together with others, led to the following model of the role of hydration water in acylation of acetylcholinesterase. The accelerator, which may also be the cationic head of the natural substrate, binds to the anionic site of the enzyme and reduces the hydration of the nucleophilic serine -OH in the esteratic site, thus enhancing the nucleophilicity of -OH. This results in an improvement of the binding between the acylating agent and the esteratic site of acetylcholinesterase.

Multiple binding of D-tubocurarine to acetylcholinesterase.

The binding of D-tubocurarine (TC) to acetylcholinesterase (AChE) was studied using different methods of enzyme kinetics. The main results are as follows. TC reversibly inhibits the hydrolysis of different substrates of AChE with three different inhibition constants (Ki1 = 7.0 +/- 0.8 X 10(-5) M, Ki2 = 3.1 +/- 1.0 X 10(-4) M, and Ki3 = 4.2 +/- 0.5 X 10(-3) M). Reference inhibitors tetramethylammonium (TMA), tetraethylammonium (TEA), and decamethonium (C-10) inhibit the hydrolysis of different substrates with constants, which are the same for each individual inhibitor. These three inhibitors compete with TC in the inhibition of enzymatic hydrolysis of acetylthiocholine (ASCh); all three of them affect the noncompetitive component of the inhibition of the hydrolysis of ASCh by TC, which arises from the binding of TC to the peripheral anionic site of AChE, but TEA and C-10 affect also the competitive component of this inhibition, which arises from the binding of TC at the catalytic anionic site. TC partially inhibits the methanesulfonylation of AChE; dissociation constant for TC in this process is KA = 4.5 X 10(-4) M. All our results lead to the conclusion that TC binds to three regions on the active surface of AChE. The first region is at the peripheral anionic site; the other two regions are situated in the vicinity of the catalytic anionic site and the esteratic site.

The acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators as an electrostatic effect.

1. In order to check our hypothesis of the electrostatic nature of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, equations were solved for methane-sulfonylation with two accelerators and the reaction was studied in the presence of some single accelerators, including the sodium cation, and in the presence of two acclerators simultaneously. 2. The second-order rate constants for methanesulfonylation of the complexes between the enzyme and accelerators decamethonium, tetraethylammonium and tetramethylammonium are 90, 88 and 17 1 - mol-1 - s-1, respectively, which corresponds to a maximal acceleration of 29, 28 and 5.5 times, respectively. The dissociation constants for the binding of these accelerators to the enzyme, obtained from our acceleration experiments, are 3.7 - 10(-6), 3.2 - 10(-4) and 1.4 - 10(-3) M, respectively. These values are in good agreement with the dissociation constants of these ligands as inhibitors of acetylcholinesterase. It is interesting to note that the sodium cation also accelerates the methane-sulfonylation up to around three times, the corresponding second-order rate constant and the dissociation constant being 10 1 - mol-1 - s-1 and 1.3 M, respectively. 3. All tested cations compete in the acceleration with each other; they seem to accelerate the reaction in the same way and from the same site, the catalytic anionic site. 4. These findings confirm the hypothesis of the electrostatic nature of acceleration.

On the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators.

1. In order to elucidate some features of the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, the methanesulfonylation of this enzyme by high concentrations of methanesulfonylfluoride, in the absence and presence of accelerators decamethonium and tetraethylammonium, was studied. 2. The results showed that the accelerator accelerates the reaction by electrostatically improving the binding between acetylcholinesterase and methanesulfonylfluoride without effecting the rate of the decomposition of the enzyme-inhibitor complex into the methanesulfonylated enzyme and product.