Competitive irreversible inhibition of enzymes in the presence of a substrate: scope and limitations

Rapid irreversible inhibition of enzymes constitutes a difficult problem and demands sophisticated techniques to meet contemporary expectations of accuracy and precision. Modern computerized, analytical techniques now allow inhibition to be measured in the presence of a chromogenic substrate, the decomposition product of which can be followed by a conventional method and in a continuous mode. This article has been written to fulfill a need for guidelines to aid the designer of experiments for the irreversible inhibition of enzymes. Thus the scope and limitations of the continuous competitive method for the irreversible inhibition of enzymes is examined here. Examples of acetylcholinesterase inhibition by two diagonally different phosphonate inhibitors are used for illustrating accuracy and precision of the competitive irreversible inhibition technique at different levels of enzyme saturation with inhibitor and substrate.     

Substrate activation of butyrylcholinesterase and substrate inhibition of acetylcholinesterase by 3,3-dimethylbutyl-N-n-butylcarbamate and 2-trimethylsilyl-ethyl-N-n-butylcarbamate

Carbamates are used to treat Alzheimer's disease. These compounds inhibit acetylcholinesterase and butyrylcholinesterase. The goal of this work is to use the substrate analogs of butyrylcholinesterase, 3,3-dimethylbutyl-N-n-butylcarbamate (1) and 2-trimethylsilyl-ethyl-N-n-butylcarbamate (2) to probe the substrate activation mechanism of butyrylcholinesterase. Compounds 1 and 2 are characterized as the pseudo substrate inhibitors of acetylcholinesterase; however, compounds 1 and 2 are characterized as the essential activators of butyrylcholinesterase. Therefore, compounds 1 and 2 mimic the substrate in the acetylcholinesterase-catalyzed reactions, but the behavior of compounds 1 and 2 mimics the substrate activation in the butyrylcholinesterase-catalyzed reactions.     

Inactivation of chymotrypsin and human skin chymase: kinetics of time-dependent inhibition in the presence of substrate

The reaction of chymase, a chymotryptic proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with time, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] much greater than [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kinetics. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants (k2) and dissociation constants (Ki) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation (k2/Ki), the first-order reactivation rate constant (k-2), and the dissociation constant of the covalent complex (Ki*) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.     

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate.

Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/Km for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (tau max = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (tau max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E'. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mm to 1 m). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E'. Substrate binds to E and E', but only Epsilon'S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.     

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.     

3-Amidoquinuclidine derivatives: synthesis of compounds and inhibition of butyrylcholinesterase

The synthesis of racemic and enantiomerically pure 3-butanamidoquinuclidines ((+/-)-Bu, (R)-Bu and (S)-Bu), (1-3) and 3-benzamidoquinuclidines ((+/-)-Bz, (R)-Bz, and (S)-Bz), (4-6) is described. The N-quaternary derivatives, N-benzyl-3-butanamidoquinuclidinium bromides ((+/-)-BnlBu, (R)-BnlBu and (S)-BnlBu), (7-9) and N-benzyl-3-benzamidoquinuclidinium bromides ((+/-)-BnlBz, (R)-BnlBz and (S)-BnlBz), (10-12) were subsequently synthesized. The interaction of the four enantiomerically pure quaternary derivatives with horse serum butyrylcholinesterase (BChE) was tested. All tested compounds inhibited the enzyme. The best inhibitior of the enzyme was (S)-BnlBz with a K(i) = 3.7 microM. The inhibitor potency decreases in order (S)-BnlBz > (R)-BnlBz > (R)-BnlBu > (S)-BnlBu.     

Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.     

Microcalorimetric study of the inhibition of butyrylcholinesterase by carbamates

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by three carbamates (eserine, neostigmine, and rivastigmine) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5). The kinetics of carbamylation was studied in the absence or presence of the substrate, butyrylcholine, using an extension of the model described by Stojan and coworkers (FEBS Lett. 440 (1998) 85-88). The model was fitted to the data by a nonlinear regression procedure using simulated annealing followed by Marquardt's method. The affinity of the carbamates for the free enzyme increased in the order neostigmine相似文献   

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH 相似文献   

Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 °C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85-88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities (K_i = 0.123 mM and K′_i = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme-inhibitor complex. The bimolecular inhibition rate constant k_i = 2.8 × 10⁴ M⁻¹ s⁻¹ was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.     

Kinetic analysis of the inhibition of human butyrylcholinesterase with cymserine

Accompanying the gradual rise in the average age of the population of most industrialized countries is a regrettable progressive rise in the number of individuals afflicted with age-related neurodegenerative disorders, epitomized by Alzheimer's disease (AD) but, additionally, including Parkinson's disease (PD) and stroke. The primary therapeutic strategy, to date, involves the use of cholinesterases inhibitors (ChEIs) to amplify residual cholinergic activity. The enzyme, acetylcholinesterase (AChE), along with other elements of the cholinergic system is depleted in the AD brain. In contrast, however, its sister enzyme, butyrylcholinesterase (BuChE), that likewise cleaves acetylcholine (ACh), is elevated and both AChE and BuChE co-localize in high amounts with the classical pathological hallmarks of AD. The mismatch between increased brain BuChE and depleted levels of both ACh and AChE, particularly late in the disease, has supported the design and development of new ChEIs with a preference for BuChE; exemplified by the novel agent, cymserine, whose binding kinetics are characterized for the first time. Specifically, as assessed by the Ellman method, cymserine demonstrated potent concentration-dependent binding with human BuChE. The IC50 was determined as 63 to 100 nM at the substrate concentration range of 25 to 800 microM BuSCh. In addition, the following new binding constants were investigated for human BuChE inhibition by cymserine: T(FPnubeta), K(nubeta), K(Bs), K(MIBA), M(IC50), D(Sc), R(f), (O)K(m), OIC100, K(sl), theta(max) and R(i). These new kinetic constants may open new avenues for the kinetic study of the inhibition of a broad array of other enzymes by a wide variety of inhibitors. In synopsis, cymserine proved to be a potent inhibitor of human BuChE in comparison to its structural analogue, phenserine.     

Cyclin-dependent kinases: inhibition and substrate recognition

Four unresolved issues of cyclin-dependent kinase (CDK) regulation have been addressed by structural studies this year - the mechanism of CDK inhibition by members of the INK4 family of CDK inhibitors, consensus substrate sequence recognition by CDKs, the role of the cyclin subunit in substrate recognition and the structural mechanism underlying CDK inhibition by phosphorylation.     

Mechanism of substrate inhibition in cellulose synergistic degradation.

A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.     

Irreversible inhibition of trypsin by TLCK. A continuous method for kinetic study of irreversible enzymatic inhibitors in the presence of substrate

This work presents a kinetic study on irreversible inhibition of trypsin by TLCK, using a new experimental approach. The method consists of the incubation of the enzyme with an irreversible inhibitor in the presence of a substrate which allows enzyme turnover as well as continuous measurement of the appearance of the product, a simultaneous change in the initial concentrations of the irreversible inhibitor and enzyme being undertaken, though a constant ratio between the latter, is maintained. This new approach enables the kinetic constants for TLCK, k2 and K1, to be determined.