Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998     

The role of electrostatic interactions in the absorption of ligands to the active sites of cholinesterases,as indicated by molecular modeling data

The effect of electrostatic interactions on the absorption of the positively charged reversible inhibitor tetram-ethylammonium, its neutral structural analogue neopentane C(CH₃)₄, and the natural substrate acethylcholine to the active sites of acetylcholinesterase and butyrylcholinesterase has been studied by molecular modeling methods. It has been shown that the dominant absorption of positively charged ligands is due to the anchoring of the cationic group of the ligand in the anionic subsite of both enzymes through the interaction of the π-cation with the benzene ring of tryptophan. The correlation between the free energy of complex formation and the catalytic activity of charged tetramethylammonium has been revealed for both enzymes. It has been shown that the effective binding of the acetylcholine molecule requires the additional activation of the enzyme.     

Long Route or Shortcut? A Molecular Dynamics Study of Traffic of Thiocholine within the Active-Site Gorge of Acetylcholinesterase

The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.     

The dynamics of ligand barrier crossing inside the acetylcholinesterase gorge

The dynamics of ligand movement through the constricted region of the acetylcholinesterase gorge is important in understanding how the ligand gains access to and is released from the active site of the enzyme. Molecular dynamics simulations of the simple ligand, tetramethylammonium, crossing this bottleneck region are conducted using umbrella potential sampling and activated flux techniques. The low potential of mean force obtained is consistent with the fast reaction rate of acetylcholinesterase observed experimentally. From the results of the activated dynamics simulations, local conformational fluctuations of the gorge residues and larger scale collective motions of the protein are found to correlate highly with the ligand crossing.     

Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.     

Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 Å radius centered in the enzyme monomer and separated from the protein surface by I-14 Å is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 Å radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within ±10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase. © 1996 John Wiley & Sons, Inc.     

Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site: EVIDENCE FOR OPEN AND CLOSED STATES OF A BACK DOOR CHANNEL*

The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site.     

Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cysteines

To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k(cat)/K(m)) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.     

Molecular docking study on the “back door” hypothesis for product clearance in acetylcholinesterase

Acetylcholinesterase (AChE) is one of the fastest enzymes known, even though the active site is buried inside the protein at the end of a 20-A deep narrow gorge. Among the great variety of crystal structures of this enzyme, both in the absence and presence of various ligands and proteins, the structure of a complex of AChE with the pseudo-irreversible inhibitor Mf268 is of particular interest, as it assists in the proposal of a back door for product clearance from the active site. Binding of Mf268 to AChE results in the carbamoylation of Ser200 and liberation of an eseroline-fragment as the leaving group. The crystal structure of the AChE-Mf268 complex, however, proves that eseroline has escaped from the enzyme, despite the fact that the Ser-bound inhibitor fragment blocks the gorge entrance. The existence of alternative routes other than through the gorge for product clearance has been postulated but is still controversially discussed in the literature, as an experimental proof for such a back door is still missing. We have used Monte Carlo-based molecular docking methods in order to examine possible alternative pathways that could allow eseroline to be released from the protein after being cleaved from the substrate by Ser200. Based on our results, a short channel at the bottom of the gorge seems to be the most probable back-door site, which begins at amino acid Trp84 and ends at the enzyme surface in a cavity close to amino acid Glu445. [Figure: see text].     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

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.     

Analysis of a 10-ns molecular dynamics simulation of mouse acetylcholinesterase.

A 10-ns molecular dynamics simulation of mouse acetylcholinesterase was analyzed, with special attention paid to the fluctuation in the width of the gorge and opening events of the back door. The trajectory was first verified to ensure its stability. We defined the gorge proper radius as the measure for the extent of gorge opening. We developed an expression of an inter-atom distance representative of the gorge proper radius in terms of projections on the principal components. This revealed the fact that collective motions of many scales contribute to the opening behavior of the gorge. Covariance and correlation results identified the motions of the protein backbone as the gorge opens. In the back-door region, side-chain dihedral angles that define the opening were identified.     

THE REACTION OF CHOLINE AND 3,3-DIMETHYL-1-BUTANOL WITH THE ACETYLENZYME FROM ACETYLCHOLINESTERASE

—A comparison was made of the manner in which choline chloride and 3,3-dimethyl-1-butanol react with the acetylenzyme formed during the hydrolysis of esters of acetic acid by acetylcholinesterase. Acetylcholine and acetylthiocholine were the substrates. The ratio of the formation of alkyl acetate to that of acetic acid increased linearly with the concentration of dimethylbutanol, but approached a limiting value as the concentration of choline was increased. Total enzymic activity was inhibited by choline and activated slightly by dimethylbutanol. The effects of varying ionic strength, pH and substrate concentration were examined. The effects of tetraethyl- and tetramethylammonium ions on the reaction of dimethylbutanol with the acetylenzyme were also studied. The results suggest that dimethylbutanol and choline bind to different regions of the active site. A mechanism for the reaction of choline and substrate with acetylcholinesterase is suggested.     

The role of Li+, Na+, and K+ in the ligand binding inside the human acetylcholinesterase gorge

Alkali cations can affect the catalytic efficiency of enzymes. This is particularly true when dealing with enzymes whose substrate bears a formal positive charge. Computational and biochemical approaches have been combined to shed light on the atomic aspects of the role of Li(+), Na(+), and K(+) on human acetylcholinesterase (hAChE) ligand binding. In this respect, molecular dynamics simulations and our recently developed metadynamics method were applied to study the entrance of the three cations in the gorge of hAChE, and their effect on the dynamical motion of a ligand (tetramethylammonium) from the bulk of the solvent into the deep narrow enzyme gorge. Furthermore, in order to support the theoretical results, K(M) and k(cat) for the acetylcholine hydrolysis in the presence of the three cations were evaluated by using an approach based on the Ellman's method. The combination of computational and biochemical experiments clearly showed that Li(+), Na(+), and K(+) may influence the ligand binding at the hAChE gorge.     

"Back door" opening implied by the crystal structure of a carbamoylated acetylcholinesterase

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.     

A study of the coordination shell of aluminum(III) and magnesium(II) in model protein environments: thermodynamics of the complex formation and metal exchange reactions

Al(III) toxicity in living organisms is based on competition with other metal cations. Mg(II) is one of the most affected cations, since the size similarity dominates over the charge identity. The slow ligand exchange rates for Al(III) render the ion useless as a metal ion at the active sites of enzymes and provide a mechanism by which Al(III) inhibits Mg(II) dependent biochemical processes. Al(III) cation interactions with relevant bioligands have been studied in a protein-model environment in gas and aqueous phases using density functional theory methods. The protein model consists of the metal cation bound to two chosen bioligands (functional groups of the amino acid side chains, one of them being always an acetate) and water molecules interacting with the cation to complete its first coordination shell. Analogous Mg(II) complexes are calculated and compared with the Al(III) ones. Formation energies of the complexes are calculated in both phases and magnesium/aluminum exchange energies evaluated. The effect of different dielectric media is also analyzed. The presence of an acetate ligand in the binding site is found to promote both, complex formation and metal exchange reactions. In addition, buried binding sites (with low dielectric constant) of the protein favor metal exchange, whereas fully solvated environments of high dielectric constant require the presence of two anionic ligands for metal exchange to occur.     

Temperature and pressure adaptation of the binding site of acetylcholinesterase

1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2 degrees C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding.     

A Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.     

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.     

Kinetic, equilibrium and spectroscopic studies on cation association at the active center of acetylcholinesterase: topographic distinction between trimethyl and trimethylammonium sites

This study examines the importance of electrostatic interactions on ligand association at the active center of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). The active-center serine was covalently modified with the dimensionally equivalent isosteric beta-(trimethylammonium)ethyl and 3,3-dimethylbutyl methylphosphonofluoridates. Reactivation of the 3,3-dimethylbutyl methylphosphono-conjugate by the bisquaternary mono-oxime HI-6, after accounting for the capacity for spontaneous reactivation, proceeded at a rate that was 20-fold greater than that for the cationic conjugate. Decidium, a fluorescent bisquaternary ligand that binds with its trimethylammonium moiety within the active center, exhibited affinity for the 3,3-dimethylbutyl conjugate that was within 2-fold that for the native enzyme, but 100-fold greater than for the cationic conjugate. Whereas association of n-alkyl mono- and bisquaternary ligands with the uncharged conjugate was virtually unaltered with respect to the native enzyme, the affinities of edrophonium, phenyltrimethylammonium and N-methylacridinium were reduced 100-fold for the uncharged conjugate relative to native enzyme. These results indicate that the orientations of the 3,3-dimethylbutyl and beta-(trimethylammonium)ethyl moieties with respect to the surface of the enzyme are not equivalent, that modification of the active center does not preclude cation association of active-center-selective ligands, and that aromatic cations associate at an anionic locus which is unique from that at which decidium and the n-alkyl mono- and bisquaternary cations associate. As such, the results point to the presence of a heterogeneity of cation binding sites within a circumscribed distance from the modified serine, and do not sustain the view proposed by Hasan et al. (J. Biol. Chem. 255 (1980) 3898-3904; 256, (1981) 7781-7785) that electrostatic interactions at the active center are subordinate to steric constraints imposed by a dimensionally restricted trimethyl site.