Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Br?nsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Br?nsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.     

A combinatorial library for the binuclear metal center of bacterial phosphotriesterase

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.     

Phosphorus-31 nuclear magnetic resonance detection of unexpected phosphodiesters in muscle

In the examination of intact muscles by 31P nuclear magnetic resonance spectroscopy, a number of signals have been detected in the phosphodiester region (-0.5 to 0.5 ppm) of the spectrum which could not be correlated with the known common phosphates of muscle tissue. These signals arise from perchloric acid extractable compounds with several common chemical properties, one of which is a ready solubility in nearly anhydrous ethanol solutions. A component contributing to the major resonance has been identified as glycerol-3-phosphorylcholine. This characterization is based on both 31P nuclear magnetic resonance and chromatographic data.     

Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.     

In vivo observation of polypeptide flux through the bacterial chaperonin system

The quantitative contribution of chaperonin GroEL to protein folding in E. coli was analyzed. A diverse set of newly synthesized polypeptides, predominantly between 10-55 kDa, interacts with GroEL, accounting for 10%-15% of all cytoplasmic protein under normal growth conditions, and for 30% or more upon exposure to heat stress. Most proteins leave GroEL rapidly within 10-30 s. We distinguish three classes of substrate proteins: (I) proteins with a chaperonin-independent folding pathway; (II) proteins, more than 50% of total, with an intermediate chaperonin dependence for which normally only a small fraction transits GroEL; and (III) a set of highly chaperonin-dependent proteins, many of which dissociate slowly from GroEL and probably require sequestration of aggregation-sensitive intermediates within the GroEL cavity for successful folding.     

Studies on transformation in Naegleria gruberi: effects of ions and bacterial suspensions

The presence of electolytes inhibited the transformation of Naegleria gruberi from amoeba to flagellate, the molarity required varying with the salt used, namely 80 mM NaCl, 90 mM KCl, 50 mM CaCl2 or 60 mM MgCl2. Non-electrolytes also prevented this transformation at 250 mM for either sucrose or glucose, and this is known to be an osmotic effect. That the effect of ionic solutions was different was demonstrated by varying the time at which the environemnt was changed from distilled water to salt solution. Experiments with suspensions of either living or heat-killed bacteria in distilled water, together with the supernatants obtained when bacteria were removed by centrifugation, showed that the inhibition of transformation which occurred in bacterial suspensions was not due to any factors produced by the bacteria and present in solution. It appeared that this inhibition was brought about by the physical presence of the bacteria, either living or heat-killed, and some possible interpretations of this 'contact' phenomenon are discussed.     

Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents

Organophosphates, such as parathion and paraoxon, constitute the largest class of insecticides currently used in industrialized nations. In addition, many of these compounds are known to inhibit mammalian acetylcholinesterases thereby acting as nerve agents. Consequently, organophosphate-degrading enzymes are of considerable interest in light of their ability to detoxify such compounds. Here we report the three-dimensional structure of such an enzyme, namely, phosphotriesterase, as determined by single crystal X-ray diffraction analysis to 2.1-A resolution. Crystals employed in this investigation belonged to the space group P2(1)2(1)2 with unit cell dimensions of a = 80.3 A, b = 93.4 A, and c = 44.8 A and one molecule per asymmetric unit. The structure was solved by multiple isomorphous replacement with two heavy-atom derivatives and refined to a crystallographic R factor of 18.0%. As observed in various other enzymes, the overall fold of the molecule consists of an alpha/beta barrel with eight strands of parallel beta-pleated sheet. In addition, there are two antiparallel beta-strands at the N-terminus. The molecular model of phosphotriesterase presented here provides the initial structural framework necessary toward understanding the enzyme's broad substrate specificities and its catalytic mechanism.     

Effect of phosphomonoesters, phosphodiesters, and phosphocreatine on glutamate uptake by synaptic vesicles

L-Glutamate, a major excitatory amino acid, plays an important role in learning and memory. L-Glutamate uptake into synaptic vesicles is an ATP-dependent process. Exposure of neurons to high, sustained extracellular concentrations of glutamate results in excitotoxicity. Elevated levels of phosphomonoesters (PMEs), phosphodiesters (PDEs), and phosphocreatine (PCr) have been reported in Alzheimer disease (AD). In this article, the effects of selected PMEs, PDEs, and PCr on vesicular L-[3H]glutamate uptake into isolated bovine synaptic vesicles are investigated. D-myo-Inositol-1-monophosphate (I1P), D-myo-inositol-2-monophosphate (I2P), sn-glycero-3-phosphate, (alpha-GP) and PCr significantly stimulated L-[3H]glutamate uptake into synaptic vesicles. Phosphoethanolamine (PE), phosphocholine (PC), L-phosphoserine (L-PS) sn-glycero-3-phosphocholine (GPC), and sn-glycero-3-phosphoethanolamine (GPE) had little or no effect on vesicular L-glutamate uptake. These observations suggested that the vesicular uptake of glutamate can be regulated by endogenous PMEs and PCr. The mechanism of activation by I1P, I2P, and alpha-GP appears to be stimulation of Mg(2+)-ATPase activity. These effects on vesicular glutamate uptake may be important in diseases in which the levels of these metabolites are altered, as they are in AD.     

Strategies to improve plant resistance to bacterial diseases through genetic engineering

Many different genetic strategies have been proposed to engineer plant resistance to bacterial diseases, including producing antibacterial proteins of non-plant origin, inhibiting bacterial pathogenicity or virulence factors, enhancing natural plant defenses and artificially inducing programmed cell death at the site of infection. These are based on our knowledge of the mechanisms of action of antibacterial compounds and of the successive steps in plant-bacterial interactions. This article presents the different approaches and demonstrates that, even though several of these ideas have already been applied, no commercial applications have yet been achieved.     

大豆蛋白复合酶解法研究

[目的]研究大豆蛋白复合酶解法的最佳工艺条件.[方法]以大豆粉为原料,采用复合酶解法研究了大豆蛋白水解度随酶解时间、加酶量、复合酶加酶间隔时间等的变化规律.[结果]大豆蛋白复合酶解法的最佳工艺条件为温度45℃,pH 7.5,液固比7:1,加酶量各0.2 g,反应时间8 h,间隔加酶时间3 h.[结论]此工艺对大豆蛋白的工业化生产具有积极的参考价值.     

酶解制备水溶性大豆多糖的研究

[目的]采用植物复合水解酶水解不溶性豆渣粉制备水溶性大豆多糖(SSPS),研究最佳制备工艺.[方法]用3,5-二硝基水杨酸比色法测定总糖和还原糖,通过单因素试验研究料液比、pH、温度、酶添加量和水解时间对SSPS得率的影响,在单因素试验的基础上设计正交试验选出优化的因素组合.[结果]各因素对SSPS得率的影响大小依次为水解温度>水解时间>酶添加量>pH,在料液比1:20(g:ml),酶添加量1.2%,pH4.5,温度50℃、提取时间25h的最佳条件下,SSPS得率为8.89%.[结论]酶解制备SSPS工艺设备简单,条件温和,但是耗时较长.     

Microstructure evolution through the α→γ phase transformation in a Ti-48 At. pct Al alloy

The α→γ phase transformation during rapid quenching and subsequent isothermal aging has been investigated in a Ti-48 at pct Al alloy. The microstructure changes from a completely massively transformed γ-grain structure to a mixed microstructure of the massively transformed γ grains and the untransformed (meaning massively untransformed) fine α ₂/γ lamellae with an increase in the cooling rate from the high-temperature α phase field. Fine γ grains are generated from these fine α ₂/γ lamellae by subsequent again at 1323 K. The fine γ grains contain many defects, such as dislocations, microtwins (or stacking faults), domain boundaries, and variants, which are frequently observed in the massive γ grains. This result suggests that the formation mechanism of the fine γ grains during aging is similar to that of the massive γ grains. When the fine γ/γ lamellar sample, which is formed by preliminary aging at a lower temperature (1173 K), is aged at a higher temperature (1323 K), apparent changes in microstructure could not be recognized. This result indicates that the fine γ-grain formation is closely related to the α ₂ → γ phase transformation in the fine α ₂/γ lamellae. This article is based on a presentation made in the symposium “Fundamentals of Gamma Titanium Aluminides,” presented at the TMS Annual Meeting, February 10–12, 1997, Orlando, Florida, under the auspices of the ASM/MSD Flow & Fracture and Phase Transformation Committees.     

稻草秸秆硫酸水解研究

[目的]优化稻草秸秆浓硫酸水解法的条件.[方法]在单因素试验的基础上,固定稻草秸秆粒度为20 ～ 40 目,以液固比(V/W)、硫酸质量分数、反应温度和反应时间4个因素进行正交试验.[结果]4个因素对稻草秸秆水解率的影响程度为:硫酸质量分数＞液固比＞反应温度＞反应时间.稻草秸秆的最佳水解条件为:硫酸质量分数70%,液固比(V/W)12:1,反应温度70 ℃,反应时间为3 h.在此条件下,稻草秸秆水解率达77%以上.[结论]该研究为综合开发利用稻草秸秆奠定了基础.     

Thermodynamics of the massive transformation

The thermodynamic nature of the massive transformation may be revealed by finding the limiting conditions for the massive mode of growth. A review of the experimental information available indicates strongly that the solvus line rather than theT ₀ line is the natural limit. Some observations of massive transformation inside the two-phase field may be explained by the effect of coherency strains in the composition spike formed in front of the interface. This spike has a significant thickness in many cases and can explain the role of the solvus. It is exceedingly thin in iron-base alloys, and the role of the solvus may there be explained by higher diffusivity inside the interface. A comparison is made with the transformation of γ in Fe-M-C alloys where the situation is very similar. This paper is based upon a presentation made at a symposium on The Massive Transformation, held at the Pittsburgh meeting of The Metallurgical Society of AIME and the Materials Science Division of ASM, October 9, 1980, under the sponsorship of the MSD Phase Transformations Committee.     

Mechanisms of the massive transformation

A massive transformation (MT) is here defined as a diffusional nucleation and growth process in which the product phase has a different crystal structure from but the same composition as the matrix phase. Particularly at low undercoolings below T _o, nucleation of a MT at a grain boundary requires that the critical nuclei be as coherent as possible with both matrix grains. This is especially important when the MT takes place in a two-phase region; that this can occur is now well documented for Ti-Al and Fe-Ni alloys. Coherency with the matrix grain toward which the nucleus is irrationally oriented, and partial coherency during growth, may be achieved through the matching of plane edges (rather than plane faces), as recently pioneered by Zhang and Kelly; Nie and Muddle; and Howe, Reynolds and Vasudevan. Massive growth requires only that diffusional jumps across massive:matrix boundaries be readily feasible at some areas of these boundaries. Although MT products usually have a “massive” appearance, this no longer appears to be an essential characteristic of this transformation mode. This article is based on a presentation made at the symposium entitled “The Mechanisms of the Massive Transformation,” a part of the Fall 2000 TMS Meeting held October 16–19, 2000, in St. Louis, Missouri, under the auspices of the ASM Phase Transformations Committee.     

水解法制备纳米二氧化钛

以工业TiOSO4为原料,采用水解法制备了纳米二氧化钛粉体,并用XHD、SEM、激光粒径分析仪对其形貌、晶型、粒径分布进行了表征。试验表明：晶种的质量对于诱导TiOSO4水解成核和控制偏钛酸的原生粒子大小有较大影响;在偏钛酸粒子沉淀中加入不同的促进剂,煅烧后．可以得到锐钛型和金红石型的二氧化钛粉体,以满足市场的不同需求。     

On the massive transformation

The massive transformation γ → α in binary FeC alloys is explored by means of a model taking into account the thermodynamic properties of the α and γ phases. In addition the model takes into account the finite mobility of the phase interface and the solute drag, i.e. the dissipation of the driving force caused by a finite solute gradient over the interphase. It is found that inside a region bounded by the α/α + γ phase boundary of the FeC phase diagram and a critical line below the T₀ line there are two solutions to the growth equations. One solution corresponds to a high growth rate controlled mainly by the finite mobility of the phase interface and corresponding to a weak solute drag. The other solution represents a lower growth rate controlled mainly by a strong solute drag. As the carbon content of the austenite is increased at a constant temperature the two solutions move closer to each other and at a critical composition before the T₀ line is reached they coincide and beyond that no massive transformation is possible. The position of the critical composition depends on L/M, the ratio of the solute diffusivity, inside the interphase, and the phase interface mobility. Inside the α one-phase region there is only one solution corresponding to a high growth rate and a weak solute drag.