A quantum-theoretic approach to enzyme specificity: Proposals for a new type of biochemical control mechanism

A quantum-theoretic approach to the problem of enzymic specificity is presented. The concept of a “measuring system” analogized with the enzyme is utilized. Along these lines a quantum mechanical hypothesis for the mechanism of enzyme reactions was advanced (Enzymologia,35, 117–130, 1968). In the measuring process an ℐ-observable, linked to the proper values of the substrate'sA ₁...A _m observables will indicate the state of the measuring apparatus. On the corresponding Hilbert space of the enzymesubstrate complexH _ES , through the respective statistical operatorU _ES we get a “state” vector [s, a]. Theng:Γ _S xΓ _E →Γ _ES , that is, to an oriented pair 〈s, a〉 ∈Γ _S xΓ _E will correspond a “state” vector [s, a], and to a proper valuei _k of ℐ will correspond, throughg, the respective equivalence classes of Г-spaces. Introducing the concept of enzyme-substrate complex entropyL _ES , a theorem is demonstrated asserting thatL _S =L _ES -L _E + 1/2kn wheren is the number of the degrees of freedom which may fluctuate. The values ofL _ES are denoted “specific values,” and it is demonstrated that a microphysical systemS may be a substrate specific forE, if and only if it can realize one of theL _ES specific values. Along these lines a model of a stochastic process for the enzymic reaction is constructed, and the set of Kolmogorov equations for the respective probabilities is derived. When a “perturbation” is induced in our model, an interesting prediction concerning the fluctuations in the kinetics of the corresponding enzyme is obtained. A relevant experimental proof ensued from these theoretical considerations. When a “gentle perturbation” was induced in a substrate by mild X-ray or UV-irradiations, an interesting oscillatory behavior of enzymic activity was recorded. A biochemical control mechanism is constructed (a simple “flip-flop” type) utilizing nonoverlapping oscillations in the activity of two enzymes at a key metabolic intermediate level. Eventually, a relevant experimental proof for the respective control model is presented.     

The smallest value of the axon density for which ‘ignition’ can occur in a random net

As shown by A. Rapoport (1952), when a very brief stimulation or “instantaneous input” is applied to a random net, the subsequent events are determined by the parameters of the net as follows: If the axon densitya is sufficiently large and the fraction γ of the neurons initially stimulated exceeds a certain value γ₁ (theover-all threshold of the net for instantaneous stimulation), excitation will spread through the net until a steady state is reached in which a fraction γ₂ ⩾ γ₁ of the neurons is firing (“ignition phenomenon”). If γ < γ₁ the activity in the net dies out. However, if the axon density is too small, the activity will ultimately die out, no matter how large the fraction of initially stimulated neurons. Thus there exists a limiting valueA of the axon density below which the net cannot “ignite”. ThisA is a function ofh, theindividual threshold of the neurons constituting the net (we assume hereh≥2, since forh=1 the situation is essentially different). Geometrically γ₁ and γ₂ are determined as the two intersection points of a straight line with a sigmoid curve. Whena<A the two curves do not intersect and fora=A they are tangent. In this paper the “tangency case” is investigated and the general features of the functionA(h) are determined. It is shown thatA increases monotonically withh (as one would expect). For all values ofh>1 we haveA(h)>h, but the fractionA(h)/h and the derivativedA(h)/dh approach unity ash increases. An analytical expression of the functionA(h) valid for very large values ofh is derived.     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Kinetic characterization of enhanced lipase activity on oil bodies

Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K _m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil substrate (activity ratio of 5:3) when compared to the native enzyme.     

pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

A “Nonsterile” Method for Selecting and Growing <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Transformants (T<Subscript>2</Subscript> Transgenic Lines) Resistant to Kanamycin

A breakthrough in transgenic Arabidopsis thaliana research was the development of the floral dip transformation protocol, a simple and reliable method of obtaining transformants, T₁ transgenic lines, at high efficiency while avoiding the use of tissue culture. However, the traditional protocol (a “sterile” method) of obtaining T₂ transgenic lines has not evolved along with improvements in transformation technology as it continues to be laborious and time-consuming. In this study, we report on the development of an improved protocol (a “nonsterile” method) for selecting and growing A. thaliana transformants (T₂ transgenic lines) resistant to kanamycin under nonsterile conditions. This protocol involves the use of a simple yet specialized device that will aid in solium selection of T₂ transgenic lines that can be rapidly grown in a hydroponic system. The “nonsterile” method reduces labor and time involved as compared to the “sterile” method; moreover, it is easy to set up and maintain. This method may also be applicable to other selecting agents, and perhaps to other plants.     

GABAA- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis

To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either “active” or “silent” preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABA_A receptors, excited silent cells and reversed GABA-induced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABA_A receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs.     

Life and death near a windy oasis

We propose a simple experiment to study delocalization and extinction in inhomogeneous biological systems. The nonlinear steady state for, say, a bacteria colony living on and near a patch of nutrient or favorable illumination (“oasis”) in the presence of a drift term (“wind”) is computed. The bacteria, described by a simple generalization of the Fisher equation, diffuse, divide A→A + A, die A→ 0, and annihilate A + A→ 0. At high wind velocities all bacteria are blown into an unfavorable region (“desert”), and the colony dies out. At low velocity a steady state concentration survives near the oasis. In between these two regimes there is a critical velocity at which bacteria first survive. If the “desert” supports a small nonzero population, this extinction transition is replaced by a delocalization transition with increasing velocity. Predictions for the behavior as a function of wind velocity are made for one and two dimensions. Received: 3 August 1998 / Revised version: 17 July 1999 / Published online: 4 July 2000     

Contribution of GABAA receptor-mediated inhibition to the determination of the velocity tuning of low pass-tuned neurons of the Superior Colliculus

The participation of intrinsic inhibitory networks in providing the velocity selectivity of neurons of the superior colliculus (SC) of the Syrian hamster was tested using iontophoretic application of bicuculline methiodide, a GABA_A receptor competitive antagonist. The impulse activity of 22 low pass-tuned (LP) cells was recorded extracellularly. Following application of bicuculline, 10 cells exhibited an increase in the velocity selectivity, while the other 12 units showed decreases in their tuning. We assume that SC intrinsic inhibitory networks contributing to the velocity tuning of neurons of this structure are driven in a dissimilar way by afferent volleys arriving from the retina through “fast” Y and “slow” W channels. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 385–387, July–October, 2007.     

Overexpression and biochemical characterization of DagA from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A ₅₄₀ = 4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A ₅₄₀ = 0.24 and 0.12, respectively) than the control (A ₅₄₀ = 0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pH 7.0. The K _m, V _max, and K _cat values for agarose were 2.18 mg/ml (approximately 1.82 × 10⁻⁵ M), 39.06 U/mg of protein, and 9.5 × 10³/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.     

Influence of Acidic Exopolysaccharide of Xanthomonas campestris IBPM 124 on the Kinetic Parameters of Extracellular Bacteriolytic Enzymes

Interactions of a negatively charged exopolysaccharide of Xanthomonas campestris IBPM 124 with its extracellular enzymes (muramidase, endopeptidase, and neutral phosphatase) and also with egg lysozyme, lysostaphin, muramidase of Streptomyces globisporus, and a bacteriolytic enzyme complex of Streptomyces albus were studied. All these enzymes were positively charged under the conditions of their maximal activity. It was shown that interaction of the acidic exopolysaccharide from X. campestris with these enzymes changed their kinetic parameters. The change was either positive (increase in reaction rate) or negative (decrease in reaction rate) and depended on the enzyme and type of substrate cleaved. Due to such interactions, the acidic exopolysaccharide secreted by X. campestris into the environment not only retained and transported positively charged exoenzymes into the near-cellular space, but also regulated their activity.     

Kinetics of partly diffusion-controlled reactions

Spectrofluorimetric methods allowing an estimation of the “microviscosity” (or the microfluidity) of synthetic and natural membranes may be used if the emisive phenomenon is dependent upon the cohesion of its local environment. Of the different methods that may be proposed, the study of the reactions between electronically excited molecules A^*, which emit fluorescence, and B quenchers embedded in membranes, the rate constant of which is partly controlled by diffusion, are expected to inform about the values of diffusion coefficients of quenchers and therefore the “microviscosity” of the environment. Information may be improved when the product of the reaction between A^* and B is itself emissive (excimers, exciplexes). We propose here that a kinetic model may apply this type of reaction when a process of static quenching occurs. The use of this kinetic model in studies carried out in this area should lead to a more accurate determination of the diffusion coefficients of A^* and B and therefore of the “microviscosity.”     

Estimation of relative contribution of “mobile phycobilisome” and “energy spillover” in the light–dark induced state transition in Spirulina platensis

Previously, it was clarified that phycobilisome (PBS) mobility and energy spillover were both involved in light-to-dark induced state transitions of intact Spirulina platensis cells. In this work, by taking advantage of the characteristic fluorescence spectra of photosystem I (PSI) trimers and monomers as indicators, the relative contributions for the “mobile PBS” and “energy spillover” are quantitatively estimated by separating the fluorescence contribution of PBS mobility from that of PSI oligomeric change. Above the phase transition temperature (T _PT) of the membrane lipids, the relative proportion of the contributions is invariable with 65% of “mobile PBS” and 35% of “energy spillover”. Below T _PT, the proportion for the “mobile PBS” becomes larger under lowering temperature even reaching 95% with 5% “energy spillover” at 0°C. It is known that lower temperature leads to a further light state due to a more reduced or oxidized PQ pool. Based on the current result, it can be deduced that disequilibrium of the redox state of the PQ pool will trigger PBS movement instead of change in the PSI oligomeric state.     

Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains

Evaluating chondrocytes in situ to document the effectiveness of cartilage preservation techniques has proven exceedingly difficult. This study was conducted to determine the effectiveness of WST-1 on porcine chondrocytes in situ after cooling to −10°C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10 mm in diameter) were harvested from sexually mature pigs within 24 h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at −10°C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at −10°C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 μm slices. Representative images were digitized and green and red pixel numbers determined the percent recovery of intact cells. Mitochondrial activity (WST-1) was determined using 20 slices of 70 μm thickness per sample to obtain reliable readings using a spectrophotometer at 450 nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144 h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7 days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48 h. MIS was most accurate when “absolute” cell recovery was compared to original controls, taking into account cell density. In conclusion, WST-1 can track metabolic activity of chondrocytes in situ over time but “absolute” cell recovery determined by MISs after 48 h incubation may be the most accurate determination of the number of live chondrocytes in situ.     

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA)

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated (“light”) and a hexadeuterated (“heavy”). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the “heavy” labelled standard with the “light” acetic anhydride and the part containing the “light” labelled standard with the “heavy” acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the “light” and the “heavy” labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: The Italian Ministry of Health     

Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity

The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)₂₂₂ and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the — (θ)₂₂₂ value. Plots of — (θ)₂₂₂and enzyme activity as a function of pH showed maximal values at pH 7.4–7.5. These results suggested the prevalence of “conformational flexibility” in the structure of serine hydroxymethyltransferase.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli. Project supported by the Chinese National “863” and “973” Projects     

An analysis of the 2o and 10o field color-matching functions

The 2^o and 10^o field color-matching functions are independent: one specification is not a linear transformation of the other, even after correcting for macular pigment effects. Therefore, the “true” color-matching functions which directly describe the linear responses of the eye must be different for the two field sizes. This means that a given stimulus will, in general, have a different chromaticity depending upon the field size, regardless of the choice of any one colorimetric co-ordinate system for all field sizes. However, in spite of these chromaticity differences, a large uniform field usually appears nearly uniform. Such color uniformity implies that even though chromatic differences occur as a function of retinal position or field size, these differences are small. If this is the case, then the underling “true” color-matching functions determining the observed color-matching functions must be nearly, but not quite, identical. These differences vanish as identity between the sets of color-matching functions is approached. This property suggests a method of calculating the “true” color-matching functions. The “true” color-matching functions must approximate those obtained by minimizing the chromaticity differences between two independent sets of data. This can be done by assuming that the coefficients of transformation should be adjusted so as to produce as nearly identical chromaticities for spectrum stimuli as possible. In this paper, it is also assumed that the “true” color-matching functions have no negative values, as if they were based on actual absorption spectra. This article describes the calculation of the “true” 2^o and 10^o field color-matching functions satisfying these two conditions. For both field sizes, the maxima of the three functions are near 435, 540, and 585 mμ, after correcting for the filtering effects of the ocular media and macular pigment.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the “red-like type” of marine algae and the “green-like type” of cyanobacteria, green algae, and higher plants. We found that the “green-like type” rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a “green-like type” rubisco from thermophilic organism.     

Effects of growth retardants on gibberellin biosynthesis inGibberella fujikurai and on growth of wheat seedlings

2-chloroethylphosphonic acid, unlike chlorocholinechloride, does not suppress gibberellin biosynthesis inGibberella fujikuroi cultures, and nullifies the effect of applied gibberellin A₃ on wheat seedling growth. Presented at the International Symposium “Plant Growth Regulators” on June 18 – 22, 1984 at Liblice, Czechoslovakia.