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J R Wild

Publications and source records attributed to J R Wild.

At least 19 recordsLinked to original sources
PubMed2001

The influence of external environment fluctuations on the signal formation of microbiosensors.

This part of theoretical analysis describes the fluctuations of output signal of microbiosensors when the number of accessible molecular recognition elements (enzymes, receptors, antibodies, etc.) fluctuated under external environmental influences. The mean electric current, dispersion correlating function, as well as spectral density of output current fluctuation are analyzed, and it is shown that a comparison of theoretically calculated mean current and correlation function with experimental data allow a determination of the kinetic parameters of substrate binding reaction with the molecular recognition element of biosensor.

Biosensing Techniques↗
PubMed2000

Allosteric signal transmission involves synergy between discrete structural units of the regulatory subunit of aspartate transcarbamoylase.

Previous studies have shown that the S5' beta-strand (r93-r97) of the regulatory polypeptides of the aspartate transcarbamoylases (ATCases) from Serratia marcescens and Escherichia coli are responsible for their diverged allosteric regulatory patterns, including conversion of CTP from an inhibitor in E. coli to an activator in S. marcescens. Similarly, mutation of residues located in the interface between the allosteric and the zinc domains resulted in conversion of the ATP responses of the E. coli enzyme from activation to inhibition, suggesting that this interface not only mediates but also discriminates the allosteric responses of ATP and CTP. To further decipher the roles and the interrelationships of these regions in allosteric communication, allosteric-zinc interface mutations (Y77F and V106A) have been introduced into both the native and the S5' beta-strand chimeric backgrounds. While the significance of this interface in the allosteric regulation has been confirmed, there is no direct evidence supporting the presence of distinct pathways for the ATP and CTP signals through this interface. The analysis of the mutational effects reported here suggested that the S5' beta-strand transmits the allosteric signal by modulating the hydrophobic allosteric-zinc interface rather than disturbing the allosteric ligand binding. Intragenic suppression by substitutions in the hydrophobic interface between the allosteric and the zinc domains of the regulatory chains resulted in the partial recovery of allosteric responses in the EC:rS5'sm chimera and reduced the activation by ATP in the Sm:rS5'ec chimera. Thus, it seems that there is a synergy between these two structural units.

Adenosine Triphosphate↗
PubMed1999

Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity for a new evaluation of the common paradigm involving allosteric control of ATCase.

Allosteric Regulation↗
PubMed1999

Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified.

Adenosine Triphosphate↗
PubMed1999

Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins.

A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.

Aryldialkylphosphatase↗
PubMed1999

Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.

Biodegradation, Environmental↗
PubMed1999

Rational design of organophosphorus hydrolase for altered substrate specificities.

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a broad variety of OP neurotoxins, including chemical warfare agents and many widely used pesticides. OPH has extremely high hydrolytic efficiency with different phosphotriester and phophothiolester pesticides (k(cat) = 50-15,000 s(-1)) as well as phosphorofluorates such as DFP and the chemical warfare agents sarin and soman (k(cat) = 50-11,000 s(-1)). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl aminoethyl) methylphosphonothioate] (k(cat) = 0.3-20 s(-1)). Different metal-associated forms of the enzyme have demonstrated varying hydrolytic capabilities for each of the OP neurotoxins, and the activity of OPH (Co2+) is consistently higher than that of OPH (Zn2+) by five- to 20-fold. Protein engineering strategies have exploited these metal-induced catalytic differences, and other slight modifications to the opd gene have resulted in significant enhancement of the rates of detoxification of the thioate pesticides and chemical warfare agents. In order to develop practical applications of OPH, other experiments have focused on improvement of enzyme production, localization, stability, and shelf-life, as well as efficient catalysis of substrates of interest.

Animals↗
PubMed1999

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.

Aryldialkylphosphatase↗
PubMed1998

Temperature effects on the allosteric responses of native and chimeric aspartate transcarbamoylases.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli have distinct allosteric regulatory patterns. It has been reported that a S. marcescens chimera, SM : rS5'ec, in which five divergent residues (r93 to r97) of the regulatory polypeptide were replaced with their Escherichia coli counterparts, possessed E. coli-like regulatory characteristics. The reverse chimera EC:rS5'sm, in which the same five residues of E. coli have been replaced with their S. marcescens counterpart, lost both heterotrophic and homotropic responses. These results indicate that the r93-r97 region is critical in defining the ATCase allosteric character. Molecular modeling of the regulatory polypeptides has suggested that the replacement of the S5' beta-strand resulted in disruption of the allosteric-zinc interface. However, the structure-function relationship could be indirect, and the disruption of the interface could influence allostery by altering the global energy of the enzyme. Studies of the temperature-sensitivity of the CTP response demonstrate that it is possible to convert CTP inhibition of the SM:rS5'ec chimera at high temperature to activation below 10 degreesC. Nonetheless, the temperature response of the native S. marcescens ATCase suggests a strong entropic effect that counteracts the CTP activation. Therefore, it is suggested that the entropy component of the coupling free energy plays a significant role in the determination of both the nature and magnitude of the allosteric effect in ATCase.

Adenosine Triphosphate↗
PubMed1998

Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli.

The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP-UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101-r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increased S0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the "tongue and groove" hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface ("reg1:cat4") between regulatory and catalytic subunits.

Adenosine Triphosphate↗
PubMed1998

Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria.

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

Bacteria, Aerobic↗
PubMed1997

Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.

The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX.

Aryldialkylphosphatase↗
PubMed1997

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.

Aryldialkylphosphatase↗
PubMed1997

Conversion of the allosteric regulatory patterns of aspartate transcarbamoylase by exchange of a single beta-strand between diverged regulatory chains.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli differ in both regulatory and catalytic characteristics. Most notably, CTP stimulates the catalytic activity of the S. marcescens ATCase and CTP/UTP inhibitory synergism has been lost. These allosteric characteristics contradict the traditional logic developed from the E. coli enzyme in which CTP and UTP function together as end products of the pyrimidine pathway to allosterically control the catalytic activity. In this study, five divergent residues (r93-r97) of the regulatory polypeptide of the S. marcescens enzyme have been replaced with their E. coli counterparts. These residues correspond to the S5' beta-strand of the allosteric effector binding domain at the junction of the allosteric and zinc domains of the regulatory polypeptide. In spite of the fact that the chimeric ATCase (SM:rS5'ec) retained 455 out of 460 amino acids of the S. marcescens enzyme, it possessed characteristics similar to those of the E. coli enzyme: (1) the [Asp]0.5 decreased from 40 to 5 mM; (2) ATP activation of the enzyme was greatly reduced; (3) CTP was converted from a strong activator to a strong inhibitor; and (4) the synergistic inhibition by CTP and UTP was restored. The S5' beta-strand is located at the outer surface of a five-stranded beta-sheet of the allosteric domain, providing a potential structural mechanism defining the allostery of this enzyme.

Allosteric Regulation↗
PubMed1997

A tryptophan-2-monooxygenase based amperometric biosensor for L-tryptophan determination: use of a competitive inhibitor as a tool for selectivity increase.

A new flow-injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L-tryptophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve between 0.1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25 degrees C and stored at 8 degrees C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 microM IA, the biosensor response to L-phenylalanine decreased to 7-4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritional broth.

Biosensing Techniques↗
PubMed1996

Allosteric regulation in a family of enterobacterial aspartate transcarbamylases: intramolecular transmission of regulatory signals in chimeric enzymes.

Several enterobacterial aspartate transcarbamylases (ATCases) exhibit a [2(C3):3(r2)] quaternary structure analogous to that of the Escherichia coli enzyme. Despite their conserved quaternary structures, these enzymes present substantial differences in the co-operativity of substrate binding and in their allosteric regulation by nucleotide effectors. A comparison between different enzymatic species provides an opportunity to expand our understanding of the molecular basis of allostery in ATCase. Chimeric ATCases were constructed by exchanging subdomain regions involved in quaternary structural features, such as the r1-c4 regulatory-catalytic subunit interface analyzed in this study, in order to define the involvement of this interface in the several components of allosteric regulation. The r1-c4 interface was found to constitute an essential element for the recognition and the transmission of the ATP regulatory signal in the Serratia marcescens and the Proteus vulgaris ATCases, as it does in the E. coli ATCase. Besides, the specific amino acid composition of the C-terminal region of the regulatory chain and its interactions with the amino acid residues in the 240s loop of the catalytic chain (r1-c4 interactions) were found to modulate the amplitude of the enzyme's response to ATP. The C-terminal region of the regulatory chain did not appear to participate directly in the regulation of the three native ATCases by CTP. Even when CTP acts as an activator, as in the P. vulgaris and S. marcescens ATCases, its signal follows a route distinct from that of the general activator ATP. Synergistic inhibition by CTP and UTP was found to involve the transmission of a specific UTP signal. This signal appeared different in the various ATCases, involving the C-terminal region of the regulatory chain in the E. coli and S. marcescens ATCases but not in the P. vulgaris ATCase.

Adenosine Triphosphate↗
PubMed1996

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins.

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Biosensing Techniques↗