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Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.

Acetylcholinesterase (AChE) (3.1.1.7) is one of the best studied enzymes found in scientific literature, partly due to its physiological role in the neurotransmission process and also to the remarkably high efficiency displayed by AChE, which has a large turnover number [

Carbaryl is a carbamate organic compound and classical noncompetitive inhibitor of AChE, with widespread use as insecticide [

Carbaryl intoxication may be confirmed by assaying AChE activity. Spectroscopic assays comprise the standard approach to determine parameters from AChE activity. They are widely used in laboratories around the world. Ellman’s method [

ITC measures the heat exchange in a physical-chemical process and is used typically for binding assays; however, this technique also allows the determination of enzyme kinetics [

When comparing the initial reaction rates (v_{0}) with the initial substrate concentration, for most of the enzymes studied a rectangular hyperbole arises; those are called Michaelis-Menten (M-M) enzymes [

The present study evaluates the kinetics of AChE by ITC, which allowed the comparison of its activity with the natural (ACh) and the artificial (ATCh) substrates. To achieve better precision, the integrated form of Michaelis-Menten equation was used to determinate experimental kinetic values. Additionally, the effect of the pesticide carbaryl, a well-known inhibitor, was also assessed and compared with the conventional methods [

The enzyme acetylcholinesterase, AChE (EC. 3.1.1.7), extracted from electric-fish (Electrophorus electricus), lyophilized, was obtained from Sigma-Aldrich®. The substrates used were acetylcholine chloride (A6625) and acetylthiocholine chloride (A5626), both obtained from Sigma-Aldrich®.

The concentration of the enzyme stock solution was determined by spectrophotometric assay at 280 nm, using ^{−1}∙cm^{−1}, and the result, confirmed by colorimetric assay using Ellman’s method [_{2} 0.01 M. The buffer with the enzyme also contained 0.1 mg/L of ultrapure bovine serum albumin (Sigma-Aldrich). The experiments were performed at pH 7.4 and 37˚C (310.15 K), except when otherwise stated.

The substrates ACh and ATCh were prepared and stocked at 0.1 M, and diluted for use in Tris-HCl buffer 0.05 M, pH 7.4 at 37˚C, with MgCl 10 mM and NaCl 100 mM. Carbaryl analytical standard (Brand Sevin® 99.6%) was stored at a concentration of 10 M in a solution of methanol 12.38 M in deionized water. The final concentration of methanol was 0.1 mM in each sample, the same concentration was added to control solutions.

Assays were performed in isothermal titrating microcalorimeter VP-ITC (MicroCal® GE). Periodic calibration of the device was conducted by the author and by laboratory team using the procedure described in the equipment manual provided by GE. The default values are described in the literature [_{2} 0.01 M (pH 7.4) at a temperature of 310.15 K. Control experiments were conducted in the absence of AChE.

Experimental solutions were degassed with a vacuum pump (ThermoVac, MicroCal®) and let to reach thermal equilibrium for 5 min prior to experimental run. The heating reference was 30 μcal/s, and the stirring speed was 215 rpm. To analyze the substrate hydrolysis, sequential injections of 1, 2, 4, 6, and 8 μl, the last until the end of the assay, with an interval of 120 s between them, of 10 mM ACh. For ATCh assay, the same protocol was used.

Todd and Gómez in 2011 [

where [P] is the molar concentration of product generated, within a certain volume (V). The power variation provided by the temperature controller maintains the temperature at the desired value (310.15 K). By Rearranging Equation (1), the reaction rate can be determined knowing the change of power provided by the VP-ITC, as the rate of heat generated by the enzyme (dq/dt) was equivalent to the variation in instrumental thermal power divided by reaction enthalpy (∆_{r}H˚’) and volume [

The variation of enthalpy can be obtained from the area definite by the power curve until it returns to the baseline, after consuming the whole substrate (S_{Total}), and it is equal to the total heat of reaction [

After these steps, the values of rate and substrate concentration could be used to determine the kinetic parameters, k_{cat}, the first-order rate constant, and K_{m}, Michaelis-Menten constant, by applying to Michaelis-Menten Equation (4). It is also possible to determine the thermodynamic parameters of activation by Eyring Equation (5):

where k_{B} is the Boltzmann constant (1.3805 × 10^{−23} J/K), h the Planck’s constant (6.6256 × 10^{−34} J/s), Δ^{‡}H and Δ^{‡}S the enthalpy and entropy of activation, T the temperature and R the gas constant (8.3145 J/K mol) [

Since its formulation, it is possible to write Michaelis-Menten as a superposition of linear and logarithmic function obtained by integrating the equation [

Nonetheless, the function is implicit, with several variables, which can be solved by numerical method with the help of basic software programs [

where, t is time observed, S_{0} is the initial substrate concentration and P_{t} is the total product formed until a time t, by a certain concentration of enzyme, [Enzyme]. To a reaction with a competitive inhibitor, inhibition constant, K_{i}, can be calculated the knowing the inhibitor concentration, I, and the turnover number, k_{cat}, by Equation (7):

The calorimetry data were analyzed with Origin 9.0, and with graphs generated with Origin 9.0, GraphPad Prism 6.01 or Microsoft Office Excel 2013.

The experiments were performed in triplicate. For each result, a statistical weighed analysis was used, which took into account the errors associated with each variable. The F-test with Akaike’s information criteria (AIC) was used to verify the similarity between regressions. Normality was tested using Shapiro- Wilk test, given P-values always superior than 0.05, and Student’s t-test was used to evaluate two groups. Linear regressions had the weight 1/y^{2} adjusted for each analysis. Data are expressed as mean (±S.E.M.).

The value of the ionization enthalpy Δ_{i}H˚ was obtained from the literature [_{r}H˚’, is a sum of the enthalpy of reaction and enthalpy of ionization for a given number of moles. As a result, the value of Δ_{i}H˚ = ?12.00 (±0.66) kJ/mol was used.

The hydrolysis of ACh by AChE gives a Δ_{r}H˚= ?29.76 (±2.19) kJ/mol (_{r}H˚ = ?24.81 (±1.70) kJ/mol (

The linear analysis from Eyring equation was used 1/y^{2} as an adjusting of weight, for decreasing the sum of squares of errors, since the y-axes, ln(k_{cat}/T), carry

most experimental error than the x-axis, 1/T. The results from calorimetric assays in different temperatures could be observed in ^{‡}G˚ values exhibited by both substrates.

The experiment was conducted in different temperatures; thus, it was possible to evaluate the increase in k_{cat} value with the rising of temperature. The Student’s t-test shown a similar result for both substrate’s Δ^{‡}G˚ (p = 0.85), due to the structural resemblance they have. Nevertheless, the values for Δ^{‡}H˚ and TΔ^{‡}S˚ are significantly distinct from each other, displaying a different driven reaction for acetylcholine (

The simultaneous nonlinear regression analysis (SNLR) uses a concurrent analysis of the data adjusted for M-M equation, the result of inhibition kinetic parameters is seen in _{cat} and K_{m} values. The k_{cat} could be defined as the number of molecules converted by an enzyme per time unit, or a first-order constant, otherwise, K_{m} is the ratio among direct and inverse reaction constants [_{cat}/K_{m} is seen as a useful indicator of the relative processing power for an enzyme [_{i} [_{i} may be thought as the amount of inhibitor required to decrease the reaction; smaller this value, the inhibitor would be more effective [_{cat} values for both analyzes were statistically equal when made F-test with Akaike’s information criterion (AIC) where ACh had a probability >88.21% to be equal in all experiments and to ATCh, they had all probability >61.77% being equal.

Substrate | Δ^{‡}G˚ (kJ/mol) | Δ^{‡}H˚ (kJ/mol) | TΔ^{‡}S˚ (kJ/mol) |
---|---|---|---|

Acetylcholine | 52.02 (±0.56) | 149.03 (±3.99) | 96.02 (±4.56) |

Acetylthiocholine | 52.41 (±1.77) | 75.65 (±3.91) | 23.23 (±5.67) |

The integrated form of Michaelis-Menten equation simultaneous analyze various concentration of product formed per time directly by the initial substrate concentration after each injection. This is an iterative nonlinear analysis method and is fitting by the method of least squares, returning the k_{cat} and K_{m} values. For this, a table is made in a simple program, such as Microsoft Office Excel®, so that all values of t, S_{0}, P_{t}, and [Enzyme] are aligned in columns side by side [

From the data obtained, the values of Δ^{‡}G˚ ACh and ATCH are very similar for both groups (p = 0.8426), which is plausible since both have a structural similarity, therefore, requiring resembling chemical steps to reach the transition state. However, the replacement of an oxygen by a sulfur in ATCh reflects the difference in the enthalpy factor activation of a substrate to another (p = 0.0001), where Δ^{‡}H˚ for ACh is almost twice that observed for ATCh. This may be due to a greater enzyme specificity for the transition state to the original substrate than for the modified. Although the value of activation entropy, Δ^{‡}S˚, increases in both cases, comments on this variation must be careful, even in a general base catalysis, as occurred in the enzyme deacylation. This reaction has a transitional state with a lot of spatial freedom, although the variation of entropy in the activated state depends not only on factors intrinsic to the reaction, but also the environment in which this occurs [

Acetylcholine | Acetylthiocholine | ||||||||
---|---|---|---|---|---|---|---|---|---|

Inhibitor (μmol/L) | k_{cat} (s^{−1}) | K_{m} (μmol/L) | k_{cat}/K_{m} (10^{7} M^{−1} s ^{−1}) | K_{i} (μmol/L) | Inhibitor (μmol/L) | k_{cat} (s^{−1}) | K_{m} (μmol/L) | k_{cat}/K_{m} (10^{7} M^{−1} s^{−1}) | K_{i} (μmol/L) |

0 | 11,315.2 | 138.3 | 8.18 | 9.72 | 0 | 9050.2 | 148.2 | 6.11 | 5.86 |

12.5 | 11,137.5 | 216.4 | 5.15 | 6.35 | 8661.1 | 205.9 | 4.21 | ||

25.0 | 11,101.5 | 663.8 | 1.67 | 10.0 | 8908.2 | 222.2 | 4.01 | ||

50.0 | 11,220.8 | 914.9 | 1.23 | 12.5 | 9024.0 | 475.1 | 1.90 | ||

75.0 | 11,180.1 | 1037.9 | 1.08 | 25.0 | 9088.3 | 916.5 | 0.99 | ||

-- | -- | -- | -- | 50.0 | 9007.8 | 1087.0 | 0.83 |

The activation value of the standard enthalpy change in the working Cabib and Wilson (1956) [

The enzyme velocity value using ATCh is lower when compared with ACh, in all analyses. This possibly occurs by the fact the enzyme has more interaction with the transition state from its natural substrate than for the synthetic substrate. As already mentioned, the catalytic efficiency of an enzyme would be given by k_{cat}/K_{m}, which is the value whose second-order constant, k_{1}, assumes whether the limiting step of the reaction was the collisional frequency between substrate and enzyme. This could only be assumed when the k_{cat} value is large enough to not be considered as limiting step, and the approach given to situations where [S] is very small, the second-order constant, k_{−1}, for the enzyme- substrate complex dissociation to free enzyme and substrate is negligible.

The values from integrated M-M are similar to those obtained by nonlinear regression analysis for both substrates. However, since only one curve was evaluated in each assay for integrated M-M, the deviation observed by the experimental fluctuation cannot be taken into account.

Analyzing all the results, it can be verified a similarity between K_{m} values for both substrates in the absence of inhibitor, motivated by the similarity in affinities to the substrates by the enzyme. As K_{m} is an apparent ratio measured of enzyme-substrate complex formation ratio to substrate catalysis into product, a low K_{m} value means that a lower concentration of substrate is required to achieve maximum rate of kinetics. Similar values of K_{m} explicit similar affinities, but only part of the problem is solved by this interpretation [

The values obtained for K_{i} allow to say that the inhibition constants of carbaryl are low and similar for both substrates. This may be a reflection of how carbaryl acts as an inhibitor, entering the active site of the enzyme, interacting with the aromatic amino acids of the anionic site of the enzyme, and blocking the entry of the substrate into the enzyme’s gorge [_{i} as the amount of inhibitor required to slow down the reaction; lower that value, more effective is an inhibitor. Thus, as the specificity of AChE to ACh is large, higher inhibitor concentrations are needed to displace ACh from the active site, but with ATCh the opposite occurs, a lower concentration of inhibitor is already able to carry carbaryl out from AChE active site, when compared to ACh.

Remarkably, the values from ordinary nonlinear regression are very similar to those obtained by using the integrated M-M analysis for both substrates. In recent fields of enzymology, there is the integrated form of the classical Michaelis-Menten equation, where the solution to catalytic constants can be reached directly by the concentration of product formed as a function of time [

Although the difference observed in both substrates structure, there is a slightly change of the values of constants because of their distinct interactions with the enzyme. This is also evidenced in different K_{i} values, although very close, the values are higher for ACh, showing that more inhibitor should be necessary to move the natural substrate of the enzyme’s active site. An alternative analysis that can save time and return more precise values is the integrated form of the Michaelis-Menten, although in this work, the implicit form was used, requiring more robust computations. The values obtained by this analysis are comparable with those obtained by classical analyses of open explicit form of the equation. Thus, the robustness of ITC enlightens an enzymatic kinetics and thermodynamic parameters values of a reaction, and even better with a more precise mathematical method. Although the traditional spectrophotometric assay is more viable to be used, this work shows comparable kinetics values between ACh and ATCh, while not precisely identical, and any approximation even if accompanied by an error is correct.

This study is supported by Brazilian Ministry of Health (n. 17217.9850001/12-025). The authors posthumously thank Professor Marcelo M. Santoro for his immeasurable help in this work.

de Almeida Neves, P.A.A., Silva, E.N. and Beirão, P.S.L. (2017) Microcalorimetric Study of Acetylcholine and Acetylthiocholine Hydrolysis by Acetylcholinesterase. Advances in Enzyme Research, 5, 1-12. https://doi.org/10.4236/aer.2017.51001