Kinetics Of Enzymatic Reaction Research Articles

Comparative activities of glutathione- S-transferase and dialdehyde reductase toward aflatoxin B1 in livers of experimental and farm animals

In order to gain a better understanding of the relative activities of glutathione- S-transferase (GST) and aldehyde reductase toward aflatoxin B1 (AFB1) in relation to the variation of species susceptibilities, we studied the in vitro cytosolic GST and reductase activities in liver tissues from male Fischer rats, ICR mice and golden hamsters, adult male rainbow trouts and female piglets. The GST activity was determined by incubating the liver cytosol with glutathione (GSH) and AFB1 in the presence of the hamster liver microsomes to metabolize AFB1 to AFB1-8, 9-epoxide. The reaction product, AFB1 and GSH conjugate (AFB1-GSH), was quantified with HPLC. The reductase activity was determined by incubating liver cytosol with AFB1-dialdehyde, followed by the quantification of the metabolic product, AFB1-dialcohol, with HPLC. All the animal species possessed the GST activities, and AFB1–GSH formed increasingly with the increase of the AFB1 concentration according to the model of first-order enzyme reaction kinetics. The V max and K m values of the GST activities in rodent species were higher and lower, respectively, than those in the trout and pig, being consistent with the relative susceptibilities to AFB1 of these animal species. However, no relationship was noted between the reductase activity and species susceptibility. Thus, the result of this study shows that GST toward AFB1, but not aldehyde reductase, is a determinant of the variation of species susceptibilities.

Kinetic Parameters of Urease Immobilized on Modified Acrylonitrile Copolymer Membranes in the Presence and Absence of Cu(II) Ions

Poly(acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications and the amount of the newly formed groups was measured for each membrane. Urease was then covalently immobilized onto the modified membranes and the amount of bound protein was determined. The kinetic parameters V(max) and K(m) of the immobilized urease were studied under static and dynamic conditions. Results showed that the rate of the enzyme reaction was higher for the membranes modified with NH(2)OH . H(2)SO(4), NH(2)NH(2) . H(2)SO(4), NaOH + EDA and NaOH + GA + EDA. It was confirmed that the reaction rate, measured under dynamic conditions, was higher than that one determined under static conditions. The influence of Cu(II) ions, as inhibitors, on the enzyme reaction kinetics (V(i) and K(i)) was also investigated. It turned out that the most sensitive membranes towards Cu(II) were those modified with NH(2)NH(2) . H(2)SO(4), NaOH + EDA and H(2)O(2). The results initiated further investigations on the influence of other heavy metal ions (Cd(II), Zn(II), Ni(II) and Pb(II)) over urease bound to a NH(2)OH . H(2)SO(4)-modified membrane. It was found that the inhibition effect of the heavy metal ions over immobilized urease decreases in the order: Cu(II) > Cd(II) > Zn(II) > Ni(II) > Pb(II). [Diagram: see text]

Surface Enzyme Kinetics for Biopolymer Microarrays: a Combination of Langmuir and Michaelis−Menten Concepts

Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] >> k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.

Kinetic equivalence of the subunits of liver alcohol dehydrogenase.

1 The kinetics of two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single-turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic-suicide experiments. 2 Amplitudes of the slow phase 328–330-nm absorbance changes occurring during the enzymic reduction of various para-substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects. 3 Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330-nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined. 4 It has been concluded in recent publications that an ordered ternary-complex mechanism involving equivalent sites is insufficient to account for the transient-state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non-equivalence of the enzymic sites. Subunit interactions resulting in a “half-site7rdquo; reactivity of the enzyme are definitely not at hand.

A Mathematical Model for the Branched Chain Amino Acid Biosynthetic Pathways of Escherichia coli K12

As a first step toward the elucidation of the systems biology of the model organism Escherichia coli, it was our goal to mathematically model a metabolic system of intermediate complexity, namely the well studied end product-regulated pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. This has been accomplished with the use of kMech (Yang, C.-R., Shapiro, B. E., Mjolsness, E. D., and Hatfield, G. W. (2005) Bioinformatics 21, in press), a Cellerator (Shapiro, B. E., Levchenko, A., Meyerowitz, E. M., Wold, B. J., and Mjolsness, E. D. (2003) Bioinformatics 19, 677-678) language extension that describes a suite of enzyme reaction mechanisms. Each enzyme mechanism is parsed by kMech into a set of fundamental association-dissociation reactions that are translated by Cellerator into ordinary differential equations. These ordinary differential equations are numerically solved by Mathematica. Any metabolic pathway can be simulated by stringing together appropriate kMech models and providing the physical and kinetic parameters for each enzyme in the pathway. Writing differential equations is not required. The mathematical model of branched chain amino acid biosynthesis in E. coli K12 presented here incorporates all of the forward and reverse enzyme reactions and regulatory circuits of the branched chain amino acid biosynthetic pathways, including single and multiple substrate (Ping Pong and Bi Bi) enzyme kinetic reactions, feedback inhibition (allosteric, competitive, and non-competitive) mechanisms, the channeling of metabolic flow through isozymes, the channeling of metabolic flow via transamination reactions, and active transport mechanisms. This model simulates the results of experimental measurements.

METABOLISM: INTERFACE BETWEEN INTEGRATIVE PHYSIOLOGY AND FUNCTIONAL GENOMICS

Edited by Kenneth B. Storey Wiley Press (2004) pp. 616. ISBN 0-471-41090-X (hbk) £ 76.50/ 108.40 (hbk) ![Figure][1] Metabolism is the principal and indispensable manifestation of life that supports the life-long homeostasis of organisms. It comprises a number of

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

Biosynthesis of deoxynucleoside triphosphates, dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg 2+ ion and the optimal Mg 2+ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.

Model-based optimization of a conductive matrix enzyme electrode.

A mathematical model has been developed to describe the mechanism for internal mass transfer and enzyme reaction kinetics of an amperometric conductive matrix enzyme electrode. The model is simplified and solved analytically to arrive at a representation for the response slope in the linear range as well as for the response time. This is the first time that the response time of an enzyme electrode is described by a mathematical model. Simulations give information on how the design parameters influence the performance of the electrode for a glucose oxidase catalyzed sensing reaction process. Based on this information, several designs were constructed and tested showing suitable agreement with theoretical predictions. Finally, an optimized electrode was designed and validated.

Progress curve analysis for enzyme and microbial kinetic reactions using explicit solutions based on the Lambert W function

We present a simple method for estimating kinetic parameters from progress curve analysis of biologically catalyzed reactions that reduce to forms analogous to the Michaelis–Menten equation. Specifically, the Lambert W function is used to obtain explicit, closed-form solutions to differential rate expressions that describe the dynamics of substrate depletion. The explicit nature of the new solutions greatly simplifies nonlinear estimation of the kinetic parameters since numerical techniques such as the Runge–Kutta and Newton–Raphson methods used to solve the differential and integral forms of the kinetic equations, respectively, are replaced with a simple algebraic expression. The applicability of this approach for estimating V max and K m in the Michaelis–Menten equation was verified using a combination of simulated and experimental progress curve data. For simulated data, final estimates of V max and K m were close to the actual values of 1 μM/h and 1 μM, respectively, while the standard errors for these parameter estimates were proportional to the error level in the simulated data sets. The method was also applied to hydrogen depletion experiments by mixed cultures of bacteria in activated sludge resulting in V max and K m estimates of 6.531 μM/h and 2.136 μM, respectively. The algebraic nature of this solution, coupled with its relatively high accuracy, makes it an attractive candidate for kinetic parameter estimation from progress curve data.

Mechanistic studies on enzymatic reactions by electrospray ionization MS using a capillary mixer with adjustable reaction chamber volume for time-resolved measurements.

Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a "makeup solvent" immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.

CMOS chip as luminescent sensor for biochemical reactions

We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-/spl mu/m CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen peroxide (H/sub 2/O/sub 2/) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H/sub 2/O/sub 2/ producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.

Laser assisted deposition of nanopatterned biomolecular layers

This work reports on the production of ultrathin films of biologically functional macromolecules with patterns in the nano to micrometer range and nanometric resolution. This technology is of importance in the quickly developing fields of bioelectronics and molecular electronics. The technique used to immobilize biological molecules in thin uniform films is based on pulsed laser vaporization/ionization of a frozen aqueous solution in a high vacuum vessel. Ionized molecules are then driven to the substrate surface by applied electric fields. Patterning is obtained by electron beam lithography and lift-off. AFM characterization of the films reveals highly uniform near monolayers with nanometric features. The samples are invariably biologically active; comparison with enzymatic reaction kinetics of known concentration solutions gives us a percentage of active molecules in the range of 10–30%. This is sufficient for the production of highly integrated bioelectronic devices.

Characterisation of the rhodanese enzyme in Trichoderma spp.

A biochemical characterisation of the rhodanese enzyme in different strains of Trichoderma was carried out in this study. The predominantly extracellular activity of this enzyme from Trichoderma was evaluated and the kinetics of the rhodanese enzyme reaction, including pH and temperature profiles, were assessed. Studies on the rhodanese enzyme from other systems have been compared with the results of this study. The kinetics of rhodanese in certain Fusarium strains, previously reported to degrade cyanides, were also analysed and compared to the enzyme from the Trichoderma. The rhodanese enzyme in all the Trichoderma strains demonstrated a broad pH optimum generally in the range from 8.5 to 11.5, along with a wide temperature optimum of 35–55 °C. The K m CN and V max values ranged from 7 to 16 mM and from 0.069 to 0.093 μmole ml −1 min −1 mg protein −1, respectively, between the selected strains of Trichoderma.

Energy storage in the photosynthetic electron-transport chain: An analogy with Michaelis-Menten kinetics

Simultaneous measurements of fluorescence and thermal emission have been performed by applying combined fluorescence and photoacoustic techniques on isolated thylakoids pretreated by prolonged illumination with saturating light. The traces were used to create Lineweaver-Burk type plots, proving clearly at least a formal analogy between the kinetics of the mechanisms governing fluorescence and thermal emission from isolated thylakoids and Michaelis-Menten kinetics of enzymatic reactions. Two characteristic parameters were calculated from them (energy storage and half-saturation light intensity) in order to obtain a basic, initial response of the photosynthetic apparatus functioning under photoinhibition stress.

Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics.

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space-filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.

Pre-steady-state kinetics of enzymatic reactions studied by electrospray mass spectrometry with on-line rapid-mixing techniques

This chapter focuses on recent developments that allow the direct on-line monitoring of enzymecatalyzed reactions by electrospray ionization (ESI) mass spectrometry (MS). In these experiments, an enzyme/substrate mixture is injected directly into the ESI source of the mass spectrometer, while the reaction proceeds in solution. With this approach it is possible to overcome many limitations of traditional kinetic methods. The new ESI MS-based techniques have the potential to become standard tools for studying enzymatic reactions in the pre-steady-state regime. These techniques are useful for kinetic studies in other fields of chemistry and biochemistry. The current time resolution of these techniques (tens of milliseconds) is not as good as that of conventional quench-flow and stopped-flow experiments. Certain restrictions may be encountered when choosing solvent systems that are compatible with on-line monitoring of the reaction mixture by ESI MS. Salts, detergents, and pH buffers may interfere with the electrospray process, leading to low signal intensities and the formation of adduct species. The electrospray process is compatible with pH buffers that are based on volatile components, such as acetic acid, formic acid, ammonia, or piperidine. It might be feasible to develop microdialysis systems for kinetic experiments that allow the rapid desalting of the reaction mixture immediately before it is analyzed by ESI MS.

Single-Molecule FRET

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions,which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices. More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3, 4]). Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a “donor” dye transfers energy to an “acceptor” dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 A, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induced-dipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by: where R is the distance between the donor and acceptor and R0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to synchronize or too rare to detect using ensemble FRET studies. Single-molecule FRET (smFRET) overcomes these difficulties, creating new opportunities to probe structural changes of biological molecules during biological events in real time. SmFRET is also relatively insensitive to incomplete labeling of host molecule with donor and acceptor because donor-only species simply show up as an FRET = 0 peak while acceptor-only species are excited only very weakly by the excitation light if the probes are selected with a large spectral separation. Unlike other single-molecule methodologies measuring forces (optical or magnetic tweezers and atomic force microscopy) [1], single-molecule FRET is sensitive to the internal motion or arrangement of host biological molecules in its center of mass frame; therefore it does not suffer from Brownian motion of the whole molecule. In addition, it can be applied to a much broader class of biological systems, because it does not require force-generating molecules. It is especially powerful in capturing rare, transient events and in dissecting complex multi-step processes. Since our first demonstration of single molecule FRET, there have been a number of biological applications (and see review in [4, 6]). Ever expanding list of biological systems that have been studied by single molecule FRET includes DNA rulers [7-9], Staphylococcal nuclease [10], biotin-Streptavidin [11], GCN4 peptides [12], a-tropomyosin [13], S15 binding RNA junction [14], Tetrahymena ribozyme [15], calmodulin [16], and Rep helicase (Ha et al, unpublished data). There are already excellent reviews on the existing single molecule FRET works [4, 6] and a review focused on practical issues regarding smFRET implementation is available [17].

Single-Molecule FRET

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions,which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices. More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3, 4]). Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a “donor” dye transfers energy to an “acceptor” dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 Å, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induced-dipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by: where R is the distance between the donor and acceptor and R0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to synchronize or too rare to detect using ensemble FRET studies. Single-molecule FRET (smFRET) overcomes these difficulties, creating new opportunities to probe structural changes of biological molecules during biological events in real time. SmFRET is also relatively insensitive to incomplete labeling of host molecule with donor and acceptor because donor-only species simply show up as an FRET = 0 peak while acceptor-only species are excited only very weakly by the excitation light if the probes are selected with a large spectral separation. Unlike other single-molecule methodologies measuring forces (optical or magnetic tweezers and atomic force microscopy) [1], single-molecule FRET is sensitive to the internal motion or arrangement of host biological molecules in its center of mass frame; therefore it does not suffer from Brownian motion of the whole molecule. In addition, it can be applied to a much broader class of biological systems, because it does not require force-generating molecules. It is especially powerful in capturing rare, transient events and in dissecting complex multi-step processes. Since our first demonstration of single molecule FRET, there have been a number of biological applications (and see review in [4, 6]). Ever expanding list of biological systems that have been studied by single molecule FRET includes DNA rulers [7-9], Staphylococcal nuclease [10], biotin-Streptavidin [11], GCN4 peptides [12], a-tropomyosin [13], S15 binding RNA junction [14], Tetrahymena ribozyme [15], calmodulin [16], and Rep helicase (Ha et al, unpublished data). There are already excellent reviews on the existing single molecule FRET works [4, 6] and a review focused on practical issues regarding smFRET implementation is available [17].

Controlling membrane permeability with bacterial porins: application to encapsulated enzymes

Recent achievements of membrane protein science allow easy protein modification by genetic engineering and, for some proteins, their production in large quantities. We regard these features as the basic requirements for applications of membrane proteins in materials science. Here, we demonstrate a possible application of membrane proteins, inserting porins from the outer cell wall of Escherichia coli into the walls of liposomes . Encapsulation of enzymes into liposomes or polymer nanocapsules protects them against proteases and denaturation. Functional reconstitution of porins into the capsule shell allows to control the rate and selectivity of substrate permeation, and thus to control the enzyme reaction kinetics. We suggest that this technique can prove to be useful in the area of biosensors, providing enzymatic stability while keeping the functionality or even enhancing the sensitivity by substrate preselection. Another application of this kind of stabilisation is in the field of single enzyme activity recording.