Oksana Lockridge | University of Nebraska Medical Center (original) (raw)

Papers by Oksana Lockridge

Biochemistry, 1998

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially ir... more Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although k cat /K m decreased 11-, 11-or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation

Biochemistry, 1995

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) ne... more Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 8, of both the active site Ser Or and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and aI1 other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x and 16 x s-l for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 "C. The free energy of activation for k3 was 19 kcal mol-', and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK, 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with K, values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100-and 2000-fold above the uncztalyzed hydrolysis of GB and VX, respectively, at pH 6.0,25.0 "C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase. Acetylcholinesterase (EC 3.1.1.7; AChEl) and butyrylcholinesterase (EC 3.1.1.8; BuChE) arise from distinct genes but share key features of protein structure, and both efficiently catalyze acetylcholine hydrolysis [reviewed in Chatonnet and Lockridge (1989) and Taylor and Radic (1994)l. Catalysis is believed to proceed through displacement of the choline moiety by a nucleophilic serinyl oxygen atom to form an acyl-enzyme intermediate, followed by attack of an internal water molecule to deacylate and regenerate the enzyme (Wilson, 1951; Taylor & Radic, 1994). Like "B-type" proteases (Hess, 1971; Fastrez & Fersht, 1973), the esteratic chemistry is carried out by a triad of Ser, His, and an acidic residue. Presumably as a consequence of their preferred binding of the carbonyl

Current Army National land use strategy seeks to protect current training/testing areas, and addr... more Current Army National land use strategy seeks to protect current training/testing areas, and address shortcomings with computer simulation, a strategy insufficient to completely meet future training and testing needs. The transformation of the U.S. Army and Department of Defense (DoD) will incorporate new weapons and tactics requiring more training land and more frequent joint-Service training than current fenced installations containing tracts of contiguous land can accommodate. This document investigates the need for a joint, distributed, and regional land use strategy that will facilitate the Army and DoD's ability to develop training/testing areas and large multi-service exercises within increasingly populated areas and regions. It examines capabilities that will be required to adopt this strategy and specifies the scope of research and development efforts, analyses, and studies required to fill capability gaps. The study team determined that Army requirements were not sufficiently documented at the time of the study to recommend creation of a dedicated Joint Distributed Regional Training work package using Army applied research (6.2) funds. This team recommends that ERDC use these results as a basis to work with the Army Environmental Requirements and Technology Assessments (AERTA) process to develop validated requirements upon which to base more focused work packages. DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

Structure and Function of Cholinesterases and Related Proteins, 1998

A study was undertaken to determine if abnormally low blood cholinesterase (ChE) activity, abnorm... more A study was undertaken to determine if abnormally low blood cholinesterase (ChE) activity, abnormal red cell acetylcholinesterase (RBC-AChE) pyridostigmine (PB) inhibition kinetics, and/or unusually high frequencies of the atypical phenotype of plasma butyrylcholinesterase (HS-BChE) could explain some of the symptoms exhibited by Gulf War veterans or represent a risk factor for adverse effects after pyridostigmine exposure.

Multidisciplinary Approaches to Cholinesterase Functions, 1992

Until now no genetic variant of human acetylcholinesterase has been reported. This enzyme is cons... more Until now no genetic variant of human acetylcholinesterase has been reported. This enzyme is considered essential to life and it was thought that genetic variants of acetylcholinesterase were incompatible with life. However, we have found a common polymorphism in human acetylcholinesterase, histidine 322 being changed to asparagine, in 5% of ACHE alleles of European and American populations. Furthermore, this genetic variation is associated with the YT blood group. We conclude that the YT blood group antigen is located on red blood cell acetylcholinesterase.

Biochemical Journal, 2014

Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents... more Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90–98% of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1...

Paraoxonase (PON1) in Health and Disease, 2002

Toxicology of Organophosphate & Carbamate Compounds, 2006

ABSTRACT

European Journal of Biochemistry, 2003

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes... more 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 α‐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 ≤ 5, under conditions of excess substrate activation. This low‐pH activity appears with wild‐type butyrylcholinesterase as well as with all mutants we examined: A328G, A328I, A328F, A328Y, A328W, E197Q, L286W, V288W and Y332A (residue A328 is at the bottom of the active‐site gorge, near the π‐cation‐binding site; E197 is next to the active‐site serine S198; L286 and V288 form the acyl‐binding pocket; and Y332 is a component of the peripheral anionic site). For example, the kcat value at pH 5.0 for activity in the presence of excess substrate was 32 900 ± 4400...

European Journal of Biochemistry, 2004

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by... more The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) < k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.

Protein Expression and Purification, 2007

The importance of human LL-37 in host defense and innate immunity is well appreciated as reflecte... more The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.

Journal of Neurochemistry, 2002

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tis... more We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE ϩ/ϩ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE ϩ/Ϫ mice had 50% of normal AChE activity and AChE Ϫ/Ϫ mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.

Journal of Molecular Neuroscience, 2006

Developmental Brain Research, 2002

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a... more Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE 2 / 2 mice were born alive and survived an average of 14 days. The emaciated appearance of AChE 2 / 2 mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (1 / 2) were fed 11%  high fat chow supplemented with liquid Ensure. AChE 2 / 2 pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE 2 / 2 mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.

Chemico-Biological Interactions, 2008

Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and ... more Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and organophosphates. Phase I safety trials of human BChE are currently being conducted and large-scale production of recombinant BChE is underway. Information on the real-time distribution of BChE from the injection site has not been well characterized. This study utilized the BChE nullizygote (BChE−/−) mouse and tetrameric equine BChE labeled with LI-COR ® fluorescent IRDye 800CW to track, quantify and determine the retention time of BChE in vivo following intramuscular injection. In vivo images were acquired with Xenogen's IVIS ® 200 imager and the LI-COR Odyssey ® Imaging System fitted with the MousePOD TM. Plasma and tissues were tested for BChE activity. The 2 mg of BChE spread from the injection site to heart, liver, intestine, kidneys, lungs, salivary glands, and muscle, but did not enter the brain or the skin. Fluorescence intensity in organs and BChE activity in plasma peaked on day 1. BChE activity in plasma was undetectable by day 16, at a time when there was still significant fluorescent signal and BChE activity in the liver (0.32 units/g), injected quadriceps (0.13 units/g) and in most of the organs analyzed. It is concluded that the tetrameric BChE glycoprotein of 340 kDa diffuses from the muscle injection site to blood and peripheral organs and has a longer residence time in the organs than in blood.

Chemico-Biological Interactions, 2010

Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their... more Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18 O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.

Chemical Research in Toxicology, 2011

The LD 50 for soman is 10 to 20-fold higher for a mouse than a human. The difference in susceptib... more The LD 50 for soman is 10 to 20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse, but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1−/− mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1−/− mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes, but was lethal only to ES1−/− mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1−/− mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.

Biochemical Pharmacology, 2000

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylch... more Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998

Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholine... more Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BuChE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca 2 or Mg 2 (6 100 mM) in buffer did not change the K m , but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BuChE by 5-fold. Turnover numbers were of the order of 5000^12 000 min 31 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl 2 at 25³C; K m values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with`atypical' BuChE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. K m value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin^BuChE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BuChE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BuChE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1U10 6 M 31 min 31. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.

Biochemistry, 1998

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially ir... more Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although k cat /K m decreased 11-, 11-or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation

Biochemistry, 1995

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) ne... more Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 8, of both the active site Ser Or and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and aI1 other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x and 16 x s-l for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 "C. The free energy of activation for k3 was 19 kcal mol-', and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK, 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with K, values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100-and 2000-fold above the uncztalyzed hydrolysis of GB and VX, respectively, at pH 6.0,25.0 "C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase. Acetylcholinesterase (EC 3.1.1.7; AChEl) and butyrylcholinesterase (EC 3.1.1.8; BuChE) arise from distinct genes but share key features of protein structure, and both efficiently catalyze acetylcholine hydrolysis [reviewed in Chatonnet and Lockridge (1989) and Taylor and Radic (1994)l. Catalysis is believed to proceed through displacement of the choline moiety by a nucleophilic serinyl oxygen atom to form an acyl-enzyme intermediate, followed by attack of an internal water molecule to deacylate and regenerate the enzyme (Wilson, 1951; Taylor & Radic, 1994). Like "B-type" proteases (Hess, 1971; Fastrez & Fersht, 1973), the esteratic chemistry is carried out by a triad of Ser, His, and an acidic residue. Presumably as a consequence of their preferred binding of the carbonyl

Current Army National land use strategy seeks to protect current training/testing areas, and addr... more Current Army National land use strategy seeks to protect current training/testing areas, and address shortcomings with computer simulation, a strategy insufficient to completely meet future training and testing needs. The transformation of the U.S. Army and Department of Defense (DoD) will incorporate new weapons and tactics requiring more training land and more frequent joint-Service training than current fenced installations containing tracts of contiguous land can accommodate. This document investigates the need for a joint, distributed, and regional land use strategy that will facilitate the Army and DoD's ability to develop training/testing areas and large multi-service exercises within increasingly populated areas and regions. It examines capabilities that will be required to adopt this strategy and specifies the scope of research and development efforts, analyses, and studies required to fill capability gaps. The study team determined that Army requirements were not sufficiently documented at the time of the study to recommend creation of a dedicated Joint Distributed Regional Training work package using Army applied research (6.2) funds. This team recommends that ERDC use these results as a basis to work with the Army Environmental Requirements and Technology Assessments (AERTA) process to develop validated requirements upon which to base more focused work packages. DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

Structure and Function of Cholinesterases and Related Proteins, 1998

A study was undertaken to determine if abnormally low blood cholinesterase (ChE) activity, abnorm... more A study was undertaken to determine if abnormally low blood cholinesterase (ChE) activity, abnormal red cell acetylcholinesterase (RBC-AChE) pyridostigmine (PB) inhibition kinetics, and/or unusually high frequencies of the atypical phenotype of plasma butyrylcholinesterase (HS-BChE) could explain some of the symptoms exhibited by Gulf War veterans or represent a risk factor for adverse effects after pyridostigmine exposure.

Multidisciplinary Approaches to Cholinesterase Functions, 1992

Until now no genetic variant of human acetylcholinesterase has been reported. This enzyme is cons... more Until now no genetic variant of human acetylcholinesterase has been reported. This enzyme is considered essential to life and it was thought that genetic variants of acetylcholinesterase were incompatible with life. However, we have found a common polymorphism in human acetylcholinesterase, histidine 322 being changed to asparagine, in 5% of ACHE alleles of European and American populations. Furthermore, this genetic variation is associated with the YT blood group. We conclude that the YT blood group antigen is located on red blood cell acetylcholinesterase.

Biochemical Journal, 2014

Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents... more Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90–98% of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1...

Paraoxonase (PON1) in Health and Disease, 2002

Toxicology of Organophosphate & Carbamate Compounds, 2006

ABSTRACT

European Journal of Biochemistry, 2003

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes... more 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 α‐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 ≤ 5, under conditions of excess substrate activation. This low‐pH activity appears with wild‐type butyrylcholinesterase as well as with all mutants we examined: A328G, A328I, A328F, A328Y, A328W, E197Q, L286W, V288W and Y332A (residue A328 is at the bottom of the active‐site gorge, near the π‐cation‐binding site; E197 is next to the active‐site serine S198; L286 and V288 form the acyl‐binding pocket; and Y332 is a component of the peripheral anionic site). For example, the kcat value at pH 5.0 for activity in the presence of excess substrate was 32 900 ± 4400...

European Journal of Biochemistry, 2004

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by... more The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) < k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.

Protein Expression and Purification, 2007

The importance of human LL-37 in host defense and innate immunity is well appreciated as reflecte... more The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.

Journal of Neurochemistry, 2002

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tis... more We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE ϩ/ϩ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE ϩ/Ϫ mice had 50% of normal AChE activity and AChE Ϫ/Ϫ mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.

Journal of Molecular Neuroscience, 2006

Developmental Brain Research, 2002

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a... more Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE 2 / 2 mice were born alive and survived an average of 14 days. The emaciated appearance of AChE 2 / 2 mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (1 / 2) were fed 11%  high fat chow supplemented with liquid Ensure. AChE 2 / 2 pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE 2 / 2 mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.

Chemico-Biological Interactions, 2008

Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and ... more Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and organophosphates. Phase I safety trials of human BChE are currently being conducted and large-scale production of recombinant BChE is underway. Information on the real-time distribution of BChE from the injection site has not been well characterized. This study utilized the BChE nullizygote (BChE−/−) mouse and tetrameric equine BChE labeled with LI-COR ® fluorescent IRDye 800CW to track, quantify and determine the retention time of BChE in vivo following intramuscular injection. In vivo images were acquired with Xenogen's IVIS ® 200 imager and the LI-COR Odyssey ® Imaging System fitted with the MousePOD TM. Plasma and tissues were tested for BChE activity. The 2 mg of BChE spread from the injection site to heart, liver, intestine, kidneys, lungs, salivary glands, and muscle, but did not enter the brain or the skin. Fluorescence intensity in organs and BChE activity in plasma peaked on day 1. BChE activity in plasma was undetectable by day 16, at a time when there was still significant fluorescent signal and BChE activity in the liver (0.32 units/g), injected quadriceps (0.13 units/g) and in most of the organs analyzed. It is concluded that the tetrameric BChE glycoprotein of 340 kDa diffuses from the muscle injection site to blood and peripheral organs and has a longer residence time in the organs than in blood.

Chemico-Biological Interactions, 2010

Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their... more Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18 O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.

Chemical Research in Toxicology, 2011

The LD 50 for soman is 10 to 20-fold higher for a mouse than a human. The difference in susceptib... more The LD 50 for soman is 10 to 20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse, but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1−/− mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1−/− mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes, but was lethal only to ES1−/− mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1−/− mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.

Biochemical Pharmacology, 2000

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylch... more Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998

Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholine... more Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BuChE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca 2 or Mg 2 (6 100 mM) in buffer did not change the K m , but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BuChE by 5-fold. Turnover numbers were of the order of 5000^12 000 min 31 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl 2 at 25³C; K m values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with`atypical' BuChE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. K m value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin^BuChE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BuChE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BuChE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1U10 6 M 31 min 31. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.