Nitrofuran induced mutagenesis and error prone repair in Escherichia coli

The binding of cis(c)- and trans(t)-Pt(NH₃)₂Cl₂ to DNA at platinum/DNA-nucleotide ratios (R_i) of 0.1 or less has been studied by means of radioactive ^195mPt-labeled compounds. Kinetic data are consistent with the following scheme:

At 25°C and pH 5–6 in 5 mM NaClO₄, the values for the rate constants in the above scheme for the c-isomer are k₂ = 2.2 × 10^?5 sec^?1, k₇ = 0.32 (sec M)^?1, and k₈ = 143 (sec M)^?1; for the t-isomer the values are k₂ < 0.5 × 10^?5 sec^?1 and k₇ = 0.95 (sec M)^?1. Platinum-DNA adducts do not undergo detectable exchange after 3 days at 37°C, indicating the absence of a dynamic equillibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag⁺ and H⁺ titration studies are consistent with binding at guanine N(7) for R_i < 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37°C and pH 7 in 0.15 M NaCl, 6–8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.     

Quantitative analysis of photosynthetic carbon metabolism in protoplasts and intact leaves of barley. Determination of carbon fluxes and pool sizes of metabolites in different cellular compartments

Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of ¹⁴CO₂. Using membrane filtration procedure, kinetics of ¹⁴C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing ¹⁴CO₂. To extract the quantitative information, analytical labeling functions P(t) describing the dependence of ¹⁴C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P(t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley.     

Evaluation of the efficiency of metabolism of dinoflagellate phosphorus and carbon by a planktonic ciliate

The trophic transfer of nutrients through the microbial food web is a key top-down control in aquatic ecosystems which is notoriously difficult to evaluate, particularly for planktonic protists. In this study, a sensitive dual-radioactive tracer technique was developed to simultaneously assess the ingestion rate, and carbon- and phosphorus-specific assimilation efficiencies, of the marine planktonic ciliate Strobilidium neptuni feeding on the autotrophic dinoflagellate Heterocapsa triquetra. Dinoflagellate prey were simultaneously 16 h pulse labelled with NaH¹⁴CO₃ and H₃³³PO₄ before being fed to the ciliate, and radioactive labels were traced into ciliate biomass and the experimental medium, as well as being monitored in the prey cells. Rates measured in short-term (10 min) incubations, as commonly used to estimate protist uptake of fluorescently labelled prey, were approximately 6 times higher and 3–6 times more variable than rates measured in longer 3–5 h incubations. The efficiency of accumulation of prey carbon (54±9%) by ciliates was lower than that of prey phosphorus (68±3%) suggesting that the phosphorus to carbon ratio in the ciliates was 1.3 times higher than in the labelled dinoflagellate biomass. Rates of phosphorus accumulation and release were combined to reveal that ciliates consumed 3.2±0.6 dinoflagellates cell^?1 h^?1. The assessment of carbon tracer release by ciliates was less reliable due to ¹⁴CO₂ exchange between the experimental media and air. The study concludes that the dual phosphorus–carbon radioactive tracer labelling of algal prey allowed the quantification of protist herbivory and nutrient remineralisation in laboratory experiments, thereby providing a potential technique for studying planktonic microbial trophic interactions in situ.     

Light-driven proton translocations in Halobacterium halobium

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating an electrochemical proton gradient across the cell membrane. However, the typical response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light is turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium.The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow.The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N′-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor.Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Acidic vesicles and the uptake of amines by sea urchin eggs

The radioactive amine [¹⁴C]methylamine is accumulated to a great extent by eggs, with kinetics that are dependent upon temperature (Q₁₀ = 5) and sensitive to metabolic inhibitors. Efflux of [¹⁴C]methylamine from eggs (preloaded with tracer concentrations) is increased immediately after fertilization or NH₄Cl activation. Fluorescent amines (9-aminoacridine (9AA) and acridine orange (AO)) are concentrated in small intracellular compartments, presumably vesicles. The possible role of these vesicles in the accumulation of amines by sea urchin eggs and the activation of the metabolism that ensues is discussed.     

Analysis of tracer experiments in non-conservative steady-state systems

A method is developed for finding the transfer and localization rates and the volumes ofN compartment steady-state biological systems from experimental results. It is shown that a complete solution for certain systems in which the rates and volumes remain constant and in which there is access to all compartments can be obtained by using a single radioactive tracer. The information obtainable from experiments wherein some compartments are not accessible is analyzed for mammillary and catenary systems. Conservative systems are handled as special cases in which the localization is zero while anisotropic membranes separating compartments are shown to introduce no additional mathematical difficulty whenever all compartments are accessible. The limitations on the use of this method of multi-compartment tracer analysis are briefly discussed. Research supported by the Atomic Energy Commission, Contract AT (30-1)-1551.     

The Rücker–Markov invariants of complex Bio-Systems: Applications in Parasitology and Neuroinformatics

Rücker's walk count (WC) indices are well-known topological indices (TIs) used in Chemoinformatics to quantify the molecular structure of drugs represented by a graph in Quantitative structure–activity/property relationship (QSAR/QSPR) studies. In this work, we introduce for the first time the higher-order (kth order) analogues (WC_k) of these indices using Markov chains. In addition, we report new QSPR models for large complex networks of different Bio-Systems useful in Parasitology and Neuroinformatics. The new type of QSPR models can be used for model checking to calculate numerical scores S(L_ij) for links L_ij (checking or re-evaluation of network connectivity) in large networks of all these fields. The method may be summarized as follows: (i) first, the WC_k(j) values are calculated for all jth nodes in a complex network already created; (ii) A linear discriminant analysis (LDA) is used to seek a linear equation that discriminates connected or linked (L_ij = 1) pairs of nodes experimentally confirmed from non-linked ones (L_ij = 0); (iii) The new model is validated with external series of pairs of nodes; (iv) The equation obtained is used to re-evaluate the connectivity quality of the network, connecting/disconnecting nodes based on the quality scores calculated with the new connectivity function. The linear QSPR models obtained yielded the following results in terms of overall test accuracy for re-construction of complex networks of different Bio-Systems: parasite–host networks (93.14%), NW Spain fasciolosis spreading networks (71.42/70.18%) and CoCoMac Brain Cortex co-activation network (86.40%). Thus, this work can contribute to the computational re-evaluation or model checking of connectivity (collation) in complex systems of any science field.     

Effect of glucose feeding on net transport of plasma free fatty acids

The effect of a single glucose feeding upon the net inflow and outflow transport of plasma free fatty acids (FFA) has been studied in 75 unanesthetized rats. The animals were fasted for 22 +/- 2 hr; then 50 rats were refed 2 ml of 50% glucose by gastric intubation. At 0, 10-15, and 30-35 min after glucose refeeding, the rats were injected with palmitate-1-(14)C complexed to rat serum. The tracer dose included (131)I-labeled albumin. Plasma FFA concentration, (131)I concentration, and FFA-(14)C were measured at five time intervals after injection of the tracer dose. From these data the irreversible disposal rate, or net outflow transport, and the net inflow transport of plasma FFA were calculated. Estimations were based upon a special case of a general solution for measuring net inflow and outflow transport of a circulating metabolite. The general solution is independent of the number of compartments, how they are interconnected, the number of nonradioactive inflows, and where the inflows enter the system. Net inflow = net outflow transport = 7.6 micro eq/min in the fasted state and 3.5 micro eq/min in the new steady state that is reached 30-40 min after glucose refeeding. A very slight imbalance between the rates of net inflow and outflow transport could account for the rapid fall in plasma FFA concentration that results from a single glucose feeding. Theoretical and practical problems associated with studying inflow and outflow transport by means of the technique using a single injection of racer are discussed.     

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification

Gas–liquid mass transfer is often rate‐limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k_La is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory‐scale stirred tank reactor (STR) with a highly efficient aeration system (k_La ≈ 570 h^?1). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k_La correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro‐differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady‐state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k_La since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory‐scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k_La) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell‐free and culture‐based methods of parameter identification, potentially confounding the determination of the “biological enhancement” of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology. Biotechnol. Bioeng. 2012; 109: 2997–3006. © 2012 Wiley Periodicals, Inc.     

Modification of the (Na+ + K+)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na⁺ + K⁺)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl (k_0.5 ca. 0.6 mm) or ATP (K_0.5 ca. 1 μm) in the medium protected against inactivation, whereas MgCl₂ (k_0.5 ca. 1 mm) increased inactivation. K⁺-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na⁺-dependent phosphorylation of the enzyme and Na⁺-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k_0.5 for K⁺-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k_0.5 for neither K⁺ nor Na⁺ was increased, although the apparent k_m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K⁺ at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k_m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a pK_a for the reactive enzyme groups of 7.5–8; and TNBS affected K⁺-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K⁺ at these sites.     

Picosecond fluorescence of intact and dissolved PSI-LHCI crystals

Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5 ± 2.5 ps) and rates of excitation energy transfer from LHCI to core (k_LC) and vice versa (k_CL). The ratio between these rates, R = k_CL/k_LC, appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. k_LC depends slightly but nonsystematically on detection wavelength, averaging (9.4 ± 4.9 ps)⁻¹. R ranges from 0.5 (<690 nm) to ∼1.3 above 720 nm.     

Controls on dissolved organic carbon composition and export from rice-dominated systems

Rice field outflow can contain high concentrations of dissolved organic carbon (DOC), which plays a crucial role in drinking water quality and aquatic ecosystem processes. This study examined the relationship between potential determining factors (i.e. rice area, outflow, drainwater reuse, soil properties, and time, measured as the day in the growing season) and the concentration and composition of DOC exported from 11 rice-dominated subwatersheds. Samples were collected from subwatershed inflow and outflow every 1–2 weeks from May through September 2008 and analyzed for DOC concentration, trihalomethane formation potential (THMFP), and also specific ultraviolet absorbance (SUVA₂₅₄) and the spectral slope parameter (S), which are indicators of DOC composition. Concentrations of DOC across all subwatersheds and sampling dates ranged from 1.56 to 14.43 mg L^?1 (mean = 4.32 mg L^?1). Linear mixed effects (LME) analysis indicated that DOC concentration decreased over time, and that THMFP, and DOC and THM flux, decreased over time, but increased with outflow. LME analysis of the SUVA₂₅₄ and S parameters indicated that the fraction of aromatic DOC moieties increased with time, outflow, and reuse. Additionally, apparent peaks in DOC concentrations, THMFP, and SUVA₂₅₄ coincided with the onsets of flooding and draining. Lastly, subwatersheds with outflow less than approximately 4,700 m³ ha^?1 behaved as sinks of DOC. Our findings suggest that water management factors such as outflow, reuse, and discrete irrigation events, all of which vary over the course of the growing season, were the dominant determinants of DOC concentration and composition.     

Disposition of intraperitoneally injected calcium-45 in suckling rats

The time course of the concentration of radiocalcium was studied in the serum, skeleton, pelt, muscles, and pooled internal organs of 10-day-old rats. Within 10 hours of injection, the specific activity of the tissue groups exceeded the specific activity of the serum and remained above it during the period studied (120 hours). Chemical and autoradiographic analyses showed how rapidly most of the injected Ca⁴⁵ found its way into the skeleton. A model was constructed with the assumption that the skeleton constitutes an essentially irreversible reservoir for the tracer in a multicompartment system in which the blood is the central or feeding compartment. The rate of transfer of the tracer from the soft tissue compartments to the serum was calculated from the equation See PDF for Equation in which C = concentration in serum (expressed as a series of exponential terms) C' = concentration in a given soft tissue Substitution in the integrated form of this equation yielded equations which had the major properties of the empirical equations fitted to the experimental points. The relative order of transfer constants (k'_–1) was: organs ≥ pelt > muscle.     

Caesium,potassium and ammonium distributions in different organs of tropical plants

In the present work the distribution of ions in aboveground plant parts was studied in order to establish the suitability of using radiocaesium as a tracer for the plant absorption of nutrients, such as potassium (K⁺) and ammonium (NH₄⁺). We present the results for the distributions of ¹³⁷Cs, ⁴⁰K and NH₄⁺ from four tropical plant species: lemon (Citrus aurantifolia), orange (Citrus sinensis), guava (Psidium guajava) and chili pepper (Capsicum frutescens). Activity concentrations of ¹³⁷Cs and ⁴⁰K were measured by gamma spectrometry and concentrations of free NH₄⁺ ions by a colorimetric method. Similarly to potassium and ammonium, caesium showed a high mobility within the plants, exhibiting the highest values of concentration in the growing parts of the tree (fruits, new leaves, twigs, and barks). A significant correlation between activity concentrations of ¹³⁷Cs and ⁴⁰K was observed in these tropical plants. The K/Cs discrimination ratios were approximately equal to unity in different compartments of each individual plant, suggesting that caesium could be a good tracer for ⁴⁰K in tropical woody fruit species. Despite the similarity observed for the behaviour of caesium and ammonium in the newly grown plant compartments, ¹³⁷Cs was not well correlated with NH₄⁺. Significant temporal changes in the NH₄⁺ concentrations were observed during the development of fruits, while the ¹³⁷Cs activity concentration alterations were not of great importance, indicating, therefore, that Cs⁺ and free NH₄⁺ ions could have distinct concentration ratios for each particular plant organ.     

Light dependence of the decay of the proton gradient in broken chloroplasts

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetic analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant k_L is equal to k_D + mR₀, where R₀ is the initial rate of proton uptake which is a function of light intensity, k_D is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K⁺ increases k_D without affecting m. Treatment with N,N′-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affecting k_D. Treatment with glutaraldehyde makes k_L independent of light intensity and hence m = 0. These results suggest that the light-dependent part (mR₀) of k_L is due to leakage of protons through the coupling factor (CF₁-CF₀) complex which can open or close depending on light intensity and that the light-independent part (k_D) of the decay constant k_L is due to proton leakage elsewhere.     

SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K_D value (12 nM) and the kinetic rate constants k_on (3 × 10⁵ M⁻¹s⁻¹) and k_off (4 × 10⁻³s⁻¹) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K_D value is in excellent agreement with the K_D value determined in solution by fluorescence titration.     

Gas–liquid mass transfer in an up-flow cocurrent packed-bed biofilm reactor

Gas–liquid mass transfer was investigated in an up-flow cocurrent packed-bed biofilm reactor. In aerobic processes gas–liquid mass transfer can be considered as a key operational parameter as well as in reactor scale-up. The present paper investigates the influence of the liquid phase mixing in the determination of the volumetric gas–liquid mass transfer coefficient (k_La) coefficient. Residence time distribution (RTD) experiments were performed in the reactor to determine the flow pattern of the liquid phase and to model mathematically the liquid phase mixing. The mathematical model derived from RTD experiments was used to evaluate the influence of the liquid mixing on the experimental estimation of the (k_La) in this reactor type. The methods used to estimate the k_La coefficient were: (i) dynamic gassing-out, (ii) sulphite method, and (iii) in-process estimation through biological conversion obtained in the reactor. The use of standard chemical engineering correlations to determine the k_La in this type of bioreactors is assessed. Experimental and modelling results show how relevant can be to take into consideration the liquid phase mixing in the calculations of the most-used methods for the estimation of k_La coefficient. k_La coefficient was found to be strongly heterogeneous along the reactor vertical axis. The value of the k_La coefficient for the packed-bed section ranged 0.01–0.12 s⁻¹. A preliminary correlation was established for up-flow cocurrent packed-bed biofilm reactors as a function of gas superficial velocity.     

A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K _m, k _cat, and k _cat/K _m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K _m, k _cat, and k _cat/K _m for Ac-nKRR-amc substrate were 100 μM, 0.112 s^?1, and 1120 M^?1·s^?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.