Báo cáo khoa học: Rat butyrylcholinesterase-catalysed hydrolysis of N-alkyl homologues of benzoylcholine

Rat Butyrylcholinesterase-Catalysed Hydrolysis of N-Alkyl Homologues of Benzoylcholine

Tác giả: Anna Hrabovská, Jean-Claude Debouzy, Marie-Thérese Froment, Ferdinand Devínsky, Ingrid Pauliková, Patrick Masson

Lĩnh vực: Hóa sinh, Dược học

Nội dung tài liệu: Nghiên cứu này khám phá các đặc tính xúc tác của enzyme butyrylcholinesterase (BChE) trên chuột khi thủy phân benzoylcholine (BzCh) và các dẫn xuất N-alkyl của nó. Các thí nghiệm cho thấy hành vi phức tạp với pha cảm ứng chậm, có dao động tắt dần (hysteresis), đặc biệt rõ rệt với các chất nền có chuỗi alkyl dài hơn. Các giá trị Km giảm khi độ dài chuỗi alkyl tăng, cho thấy ái lực cao hơn của enzyme đối với các chất nền dài hơn. Tuy nhiên, kcat lại giảm đối với các chất nền dài, do định hướng không tối ưu trong trung tâm hoạt động của enzyme. Nghiên cứu cũng sử dụng kỹ thuật NMR để phân tích cấu trúc và sự tương tác của các chất nền, cũng như mô hình hóa phân tử để dự đoán cách các chất nền liên kết với enzyme. Kết quả chỉ ra rằng BzCh và các dẫn xuất alkyl ngắn hơn (BCHn, n=2-4) là chất nền tốt cho BChE trên chuột, trong khi các dẫn xuất alkyl dài hơn lại kém hiệu quả hơn.

Mục lục chi tiết:

• Keywords
• Correspondence
• Present address
• Note
• (Received 19 September 2005, revised 6 January 2006, accepted 17 January 2006)
• doi:10.1111/j.1742-4658.2006.05144.x
• Cholinesterases (ChEs; E.C. 3.1.1) are serine hydrolases that primarily act on choline esters. Acetylcholinesterase (E.C. 3.1.1.7) primarily hydrolyses esters with short acyl moiety, like acetylcholine (ACh). Butyrylcholinesterase (E.C. 3.1.1.8) prefers esters with longer aliphatic or aromatic acyl moiety, like butyrylcholine and benzoylcholine (BzCh).
• The main function of acetylcholinesterase is to hydrolyse ACh in the central nervous system, in postganglionic synapses of the parasympathetic system and at neuromuscular junctions.
• By hydrolysis of this neurotransmitter, acetylcholinesterase regulates overexcitation of muscarinic and nicotinic cholinoceptors.
• The physiological role of butyrylcholinesterase is unknown so far.
• Based on the ability to hydrolyse ACh, it is possible that butyrylcholinesterase substitutes cholinergic function of acetylcholinesterase in the central nervous system [1].
• High butyrylcholinesterase activity in many first contact tissues (e.g. lungs, skin, blood and placenta) as well as high affinity towards a wide range of xenobiotics support the hypothesis that butyrylcholinesterase is primarily a detoxification enzyme.
• Other hypotheses about possible noncholinergic functions of ChEs outside cholinergic synapses have been reported in the literature [2].
• ChEs have been a hot topic for scientists for the past 60 years.
• It is because of their inhibition by poisonous carbamyl- and phosphoryl-esters [3-5], their participation in the metabolism of the xenobiotics (e.g. cocaine, procainamide, acetylsalicylic acid) [6-8] and correlation between their activity and pathological processes (e.g. Alzheimer disease, myasthenia gravis, Parkinson’s disease) [9-13].
• Wide use of organophosphates and carbamates in farming as well as the threat of organophosphorus warfare agents led to the study of their deleterious effects on human ChEs.
• Blocking acetylcholinesterase leads to serious physiological function impairments and damage in the nervous system, even to death [14-17].
• The active site of ChEs is located at the bottom of a 20-Å deep active gorge.
• The peripheral anionic site (PAS) is located at the entrance to the active site gorge.
• It is considered to be an initial binding site for substrates.
• There is 81% identity of rat butyrylcholinesterase with human butyrylcholinesterase at nucleotide level and 80% identity at amino acid sequence level.
• Rat butyrylcholinesterase is exactly the same length as human butyrylcholinesterase.
• This makes numbering for the two enzymes identical [18].
• Active site gorge amino acid sequence of rat butyrylcholinesterase differs from the human enzyme in eight residues.
• Four of them are conservative replacements while other four are not.
• One of them (Q223E) is situated below the catalytic triad, away from substrate binding site, other two (L286R and P285I) are situated in the acyl-binding pocket and the last one (A277K) brings the positive charge into the mouth of the gorge [18].
• The last mentioned could possibly change interaction of the enzyme with substrates compared to human butyrylcholinesterase.
• ChEs do not follow the Michaelis-Menten kinetics with positively charged substrates.
• Inhibition/activation by excess substrate depends on pH [19-21].
• Other complexities in kinetics of human butyrylcholinesterase were observed.
• Hysteresis in the approach to steady-state was observed for certain neutral and charged substrates.
• acetylcholinesterase [22] and butyrylcholinesterase [23] show slow transient kinetics with neutral ester N-methylindoxyl acetate (NMIA).
• Pre-steady-state induction phase can be explained by slow transition from a form E to a form E’ and enzyme-substrate complexes ES to E’S, while only E’S leads to product formation.
• More complex hysteresis was observed for butyrylcholinesterase when BzCh was used as substrate, where damped oscillations superimposed on the hysteretic lag [24].
• Damped oscillations were explained by the time-dependent change in substrate concentration caused by the presence of multiple substrate forms (aggregates and conformers) in slow equilibrium, and only one of these forms interacts with enzyme.
• A model for human butyrylcholinesterase-catalysed hydrolysis of BzCh was proposed:
• In this model k₀ and k₀ are the first-order rate constants for the reversible transition between E and E’.
• k_es and k_-es are the first-order rate constants for the reversible transition between ES’ and E’S’.
• k₁ and k’₁ are rate constants for substrate binding to E and E’.
• ES’ and E’S’ are the enzyme-substrate complexes.
• Only E’S’ makes product (P).
• k_s and k_-s are slow rate constants for molecular conversion of substrate forms S and S’, and k_s > k_s.
• The purpose of this work was to determine whether rat butyrylcholinesterase displays a similar behaviour in the approach to steady state with BzCh, to determine the steady-state catalytic parameters, and to investigate the catalytic behaviour of this enzyme with N-alkyl homologues of BzCh.
• Results
• Pre-steady-state
• Slow pre-steady-state was observed for hydrolysis of various neutral and charged substrates by human and horse butyrylcholinesterase.
• Like human butyrylcholinesterase, rat butyrylcholinesterase showed, despite the presence of a long lag phase with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate.
• Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active.
• The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium.
• Different substrate conformational states were confirmed by ¹H-NMR.
• The K_m values for substrates decreased as the length of the alkyl chain increased.
• High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge.
• Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased.
• The long-chain substrates had reduced k_cat values.
• Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis.
• The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V_max.
• This was due to the loss of substrate availability.
• The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh – BCH4.
• Steady state kinetics
• BzCh is a good substrate of butyrylcholinesterase.
• The kinetics of butyrylcholinesterase-catalysed hydrolysis of BzCh was described previously [18,24-28].
• Here we studied hydrolysis of N-alkyl homologues of BzCh by rat butyrylcholinesterase.
• Rat butyrylcholinesterase-catalysed hydrolysis of long chain substrates BCH14–BCH18 showed unusual behaviour.
• We observed premature plateau in the kinetics with respect to substrate concentration (marked by an asterisk in Table 1), suggesting substrate unavailability.
• We were able to follow the hydrolysis of BCH14 only at very low substrate concentration and thus we could only determine the V_max/K_m (or k_cat/K_m) ratio.
• It was not possible to follow hydrolysis of BCH16 and BCH18 even at low concentrations.
• Abnormal behaviour of long-chain substrates was confirmed by nonlinearity of the progress curves v versus t (Fig. S1).
• Therefore these homologues were excluded from the study.
• Steady-state parameters are listed in Table 1.
• The highest K_m values were observed for the shortest homologues (BzCh–BCH4) with a maximum for BzCh.
• K_m for homologues with six and more carbons in the alkyl chain dropped to one-tenth of the value for BzCh.
• This suggests very high affinity of these substrates for rat butyrylcholinesterase.
• k_cat reached the highest value for BzCh.
• It persisted at the high level for another two homologues (BCH2 and BCH4).
• Significant drop in k_cat to the extremely low level was observed for higher homologues, starting with BCH6.
• K_m and k_cat values for rat butyrylcholinesterase-catalysed BzCh hydrolysis were comparable to the ones obtained for human enzyme (K_m = 3 ± 0.3 µm and k_cat = 245 ± 7 s⁻¹ for wild-type butyrylcholinesterase and K_m = 21.1 ± 4.9 µm and k_cat = 250 ± 16 s⁻¹ for D70G mutant) [24] and the ones obtained for rat wild-type butyrylcholinesterase (K_m = 43 ± 1.6 µm and k_cat = 388 ± 13 s⁻¹) [18].
• Catalytic efficiency of rat butyrylcholinesterase expressed by k_cat/K_m is of the same order for all studied substrates.
• Deacylation rate constant k₃ determined for the human enzyme was used for calculations (k₃ = 330 s⁻¹) [24] as the same acylated intermediate was formed in hydrolysis of all studied substrates.
• Homologues could be divided into the three groups based on the values of the rate constant for the acylation step (k₂).
• The maximum was observed for BzCh, a 60% drop in k₂ value was seen for short alkyl chain substrates and up to a 97% decrease for longer chain homologues (BCH6–BCH12).
• Dissociation constant of E’S complex (K_s) depended on the N-substituted chain length and had decreasing tendency with increasing alkyl length.
• Beginning with BCH6 it persisted at one-thirtieth of the BzCh value.
• The rate-limiting step of enzyme hydrolysis was determined for each substrate based on the calculated kinetic parameters.
• The literature indicates that deacylation is partly rate-limiting step of BzCh hydrolysis by human wild-type butyrylcholinesterase.
• We observed in the case of rat butyrylcholinesterase that deacylation becomes fully rate-limiting for hydrolysis of this substrate (k₂ ≫ k₃).
• The difference between both enzymes may be explained by the differences in architecture of the active site gorge of rat butyrylcholinesterase compared to the human enzyme [18].
• We hypothesize that changes in acyl binding pocket (L286R and P285I) are crucial for the deacylation step.
• Proline 285 in human butyrylcholinesterase is replaced by isoleucine in the rat enzyme.
• Such a radical change in structure of the acyl binding pocket very likely affects the enzyme deacylation rate.
• Residue 286 defines mostly the substrate specificity.
• However, it also plays a role in deacylation.
• In particular it was observed that the reactivation rate of diethylphosphate-inhibited human butyrylcholinesterase increased after substituting histidine into position 286 [18].
• Introduction of a positive charge at the mouth of the gorge by a lysine residue at position 277 in rat butyrylcholinesterase could influence the rate-limiting step.
• A possible effect of A277K on acylation and deacylation cannot be ruled out.
• A similar shift towards full rate-limiting deacylation was observed in human butyrylcholinesterase carrying a mutation in PAS (D70G) [24].
• Limitation of hydrolytic rate was shifted toward acylation as the alkyl side chain length increased.
• Deacylation is partly rate limiting for hydrolysis of short chain homologues (BCH2, BCH4), but the rate-limiting step for homologues with longer alkyl chain is acylation, as a consequence of the k_cat value drop (k₂ < k₃).
• It was confirmed that the rate of ChE-catalysed hydrolysis of good substrates is limited by deacylation.
• ChE-catalysed hydrolysis of poor substrates is characterized by rate-limiting acylation [29,30].
• Based on that, the best rat butyrylcholinesterase substrate among studied homologues is BzCh and the worst ones are, despite their high affinity for the enzyme, the longer homologues (starting with BCH6).
• Premature plateau in the steady-state kinetics and nonlinear progress curves for the longest alkyl chain homologues, as well as damped oscillations in pre-steady state kinetics of all substrates were hypothesized to be caused by the presence of multiple molecular forms of substrate.
• To determine the actual molecular conformation of studied homologue substrates, ¹H-NMR spectra were recorded.
• ¹H-NMR study of substrate conformational polymorphism
• Experimental conditions are described in Experimental procedures.
• Chemical shifts, linewidths and relaxation times were inspected, with special attention to the three building blocks of BCHn, i.e. the aromatic ring resonances (7-9 p.p.m. region), the N⁺(CH₃)₃ choline head resonance (around 3.2 p.p.m) and the alkyl chain terminal methyl group resonance (around 0.8 p.p.m).
• Figure 2 shows the building blocks and protons whose resonances were recorded.
• Chemical shifts
• Aromatic resonance
• Figure 3A shows typical spectra recorded on the aromatic ring of several derivatives at the same concentration (5 mm).
• The chemical shift (δ) values of all samples’ aromatic rings for the extreme concentrations (10 µm and 5 mm) are also presented in Fig. 3B.
• δ characterizes spin shielding and is very sensitive to environment changes.
• No significant changes in δ were observed for homologues BCH2–BCH10 at any concentration.
• Thus, it can be concluded that no major local conformation change took place.
• By contrast, the values for BCH10 and BCH12 dropped (Δδ up to 0.3 p.p.m), revealing a modification of the local magnetic environment.
• Further decrease in δ was seen for the highest homologues (BCH14–BCH18), but only for protons on Cα and Cβ.
• This indicates that molecular re-arrangement of the aromatic ring is not homogenous.
• Choline and chain resonances
• Only minor nonsignificant δ variations were found for N-substituted choline head and N-alkyl chain methyl resonances (except for BzCh in which N-alkyl chain is trimethyl; δ = 3.23 p.p.m).
• These indicate only limited local conformation changes related to the chain length and concentration, as usually observed for alkyl chains [31].
• Linewidths and T₂
• Linewidths and transverse relaxation times are closely related to molecular motion.
• As T₂ could be