Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging

The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.     

Studying the roles of W86, E202, and Y337 in binding of acetylcholine to acetylcholinesterase using a combined molecular dynamics and multiple docking approach

A combined molecular dynamics simulation and multiple ligand docking approach is applied to study the roles of the anionic subsite residues (W86, E202, Y337) in the binding of acetylcholine (ACh) to acetylcholinesterase (AChE). We find that E202 stabilizes docking of ACh via electrostatic interactions. However, we find no significant electrostatic contribution from the aromatic residues. Docking energies of ACh to mutant AChE show a more pronounced effect because of size/shape complementarity. Mutating to smaller residues results in poorer binding, both in terms of docking energy and statistical docking probability. Besides separating out electrostatics by turning off the partial charges from each residue and comparing it with the native, the mutations in this study are W86F, W86A, E202D, E202Q, E202A, Y337F, and Y337A. We also find that all perturbations result in a significant reduction in binding of extended ACh in the catalytically productive orientation. This effect is primarily caused by a small shift in preferred position of the quaternary tail.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the π-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the π-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase?

The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine, decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5-, 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-type HuAChE. For most of these prototypical noncovalent active center and peripheral site ligands, the hexamutant HuAChE displayed a reactivity phenotype closely resembling that of HuBChE. These results support the accepted view that the active center architectures of AChE and BChE differ mainly by the presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthiocholine, or covalent ligands such as phosphonates and the transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), is about 45-170-fold lower than that of HuBChE. Most of this reduction in reactivity can be related to the combined replacements of the three aromatic residues at the active center, Phe295, Phe297, and Tyr337. We propose that the hexamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate certain tetrahedral species in the active center. This impairment may be related to the enhanced mobility of the catalytic histidine His447, which is observed in molecular dynamics simulations of the hexamutant and the F295L/F297V/Y337A HuAChE enzymes but not in the wild-type HuAChE.     

Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (k _ET) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher k _cat values than their corresponding k _ET, implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.     

Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway

Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.     

Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.     

The aromatic "trapping" of the catalytic histidine is essential for efficient catalysis in acetylcholinesterase

While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.     

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   

Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Effects of active site cleft residues on oligosaccharide binding,hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants

Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β‐glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X‐ray crystal structures of cellooligosaccharide complexes with its E176Q acid‐base and E386G nucleophile mutants. X‐ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X‐ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.     

Insights into the Functional Architecture of the Catalytic Center of a Maize β-Glucosidase Zm-p60.1

The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K(m) of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.     

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.     

Homology modelling and site-directed mutagenesis studies of the epoxide hydrolase from Phanerochaete chrysosporium

Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida.

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) [Schwede, T.F., Rétey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355-5361]. To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively. The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in catalysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator. All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.     

Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase

Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.     

Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers

The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.