Applied Photophysics - For Circular Dichroism and Stopped-flow Spectroscopy

References

The last 25 references for the SX-series are listed below. A complete searchable database with all known stopped-flow references can be accessed by logging into the APL members area.

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AuthorsTitleYearKeywordsJournal/Proceedings
Emile Bol, Nicolette J. Broers, and Wilfred R. Hagen A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus

[Abstract]   [URL]

2008 Tungsten, Formaldehyde oxidoreductase, Pyrococcus furiosus, Pre-steady-state kinetics, Steady-state kinetics J BIOL INORG CHEM, 2008, Vol 13, Iss 1, pp 75-84
Abstract: Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM-1 cm-1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s-1, kobs2 = 1.9 s-1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 × 10-2 s-1, kobs4 = 2.18 × 10-2 s-1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
Biljana Petrovi, ivadin D. Bugari and Rudi van Eldik Kinetic studies on the reactions of [Pd(dach)(X–Y)] complexes with some DNA constituents

[Abstract]   [URL]

2008 [Pd(dach)(X–Y)] , DNA, Kinetic studies J CHEM SOC DALTON TRANS, 2008
Abstract: The kinetics of complex-formation reactions of six Pd(dach) complexes, dach = 1,2-trans-R,R-diaminocyclohexane, viz. [Pd(dach)Cl2], [Pd(dach)(H2O)2]2+, and four complexes with different chelating leaving groups X–Y, viz. [Pd(dach)(O,O-cyclobutane-1,1-dicarboxylate)], [Pd(dach)(N,O-glycine)]+, [Pd(dach)(N,S-methionine)]+ and [Pd(dach)(O,O-oxalate)], were studied. The effect of the leaving group on the lability of the resulting Pd(II) complexes was studied for the nucleophiles inosine, inosine-5-monophosphate and guanosine-5-monophosphate under pseudo-first-order conditions as a function of nucleophile concentration, temperature and pressure using stopped-flow techniques. Two consecutive reaction steps, which both depend on the nucleophile concentration, were observed. The rate constants for all reactions indicate a direct substitution of the X–Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz. O–O (cbdca), (ox), N–O (gly) or S–N (L-met), plays an important role in the kinetic and mechanistic behavior of the Pd(II) complexes. The mechanism of the substitution reactions is associative in nature as supported by the large and negative values of ΔS and ΔV.
Naomi Courtemanche and Doug Barrick Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B

[Abstract]   [URL]

2008 repeat protein, leucine-rich repeat, protein folding, kinetics PROTEIN SCI, 2008, Vol 17, pp 43-53
Abstract: Although the folding of {alpha}-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing β-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single β-strand that forms a continuous β-sheet with neighboring repeats, and an N-terminal α-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in α-helical repeat proteins, to date, InlB is the only β-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.
Wolfhardt Freinbichler, Maria A. Colivicchi, Manuela Fattori, Chiara Ballini, Keith F. Tipton, Wolfgang Linert and Laura Della Corte Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

[Abstract]   [URL]

2008 Vertebrata ; Mammalia ; Rodentia ; Basal ganglion ; Excitatory aminoacid ; Central nervous system ; Toxin ; Rat ; Neurotoxin ; Fluorescence ; In vitro ; Peroxides ; Oxygen ; Sodium ; Microdialysis ; Encephalon ; Release ; Neurotransmitter ; Radical ; J NEUROCHEM, 2008
Abstract: Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA2-) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o-phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA2- in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ‘ex vivo’ showed TA2- to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2- forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.
Lucy G. Randles, Sarah Batey, Annette Steward, and Jane Clarke Distinguishing Specific and Nonspecific Interdomain Interactions in Multidomain Proteins

[Abstract]   [URL]

2008 Multidomain proteins, Interdomain Interactions, spectrin-titin BIOPHYS J, 2008, Vol 94, pp 622-628
Abstract: Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.
Hothi, P.; Lee, M.; Cullis, P. M.; Leys, D.; Scrutton, N. S. Catalysis by the Isolated Tryptophan Tryptophylquinone-Containing Subunit of Aromatic Amine Dehydrogenase Is Distinct from Native Enzyme and Synthetic Model Compounds and Allows Further Probing of TTQ Mechanism

[Abstract]   [URL]

2008 benzylamines, Aromatic Amine Dehydrogenase, TTQ Mechanism BIOCHEMISTRY-USA, 2008, Vol 47, Iss 1, pp 183-194
Abstract: Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies with TTQ-dependent aromatic amine dehydrogenase (AADH). In this study, we combine kinetic isotope effects (KIEs) with structure-reactivity studies to show that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated TTQ-containing subunit of AADH. Contrary to the TTQ-containing subunit of methylamine dehydrogenase (MADH), which is catalytically inactive, the small subunit of AADH catalyzes the oxidative deamination of a variety of amine substrates. Observed rate constants are second order with respect to substrate and inhibitor (phenylhydrazine) concentration. Kinetic studies with para-substituted benzylamines and their dideuterated counterparts reveal KIEs (>6) larger than those observed with native AADH (KIEs ~ unity). This is attributed to formation of the benzylamine-derived iminoquinone requiring structural rearrangement of the benzyl side chain in the active site of the native enzyme. This structural reorganization requires motions from the side chains of adjacent residues (which are absent in the isolated small subunit). The position of Phe97 in particular is responsible for the conformational gating (and hence deflated KIEs) observed with para-substituted benzylamines in the native enzyme. Hammett plots for the small subunit exhibit a strong correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenethylamines, unlike native AADH for which a poor correlation is observed. TTQ reduction in the isolated subunit is enhanced by electron withdrawing substituents, contrary to structure-reactivity studies reported for synthetic TTQ model compounds in which rate constants are enhanced by electron donating substituents. We infer that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated small subunit. This is attributed to the absence of structural rearrangement prior to H-transfer that limits the rate of TTQ reduction by para-substituted benzylamines in native enzyme.
Berka, V.; Wang, L.-H.; Tsai, A.-L. Oxygen-Induced Radical Intermediates in the nNOS Oxygenase Domain Regulated by L-Arginine, Tetrahydrobiopterin, and Thiol

[Abstract]   [URL]

2008 nNOS Oxygenase, nitric oxide, BIOCHEMISTRY-USA, 2008, Vol 47, Iss 1, pp 405-420
Abstract: Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), L-citrulline, NADP+, and water from L-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and L-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or L-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and L-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or L-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and L-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without L-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new ~75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only L-arginine in nNOSox (without BH4 but with L-Arg) caused conversion of ~70% of superoxide radical to a novel radical, explaining how L-arginine decreases the level of superoxide production in nNOSox (without BH4 but with L-Arg). The regulatory role of L-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.
Wang, X.; Stanbury, D. M. Direct Oxidation of L-Cysteine by [FeIII(bpy)2(CN)2]+ and [FeIII(bpy)(CN)4]-

[Abstract]   [URL]

2008 L-Cysteine, oxidation Inorg. Chem., 47 (3), 1224 -1236, 2008.
Abstract: The oxidation of L-cysteine by the outer-sphere oxidants [Fe(bpy)2(CN)2]+ and [Fe(bpy)(CN)4]- in anaerobic aqueous solution is highly susceptible to catalysis by trace amounts of copper ions. This copper catalysis is effectively inhibited with the addition of 1.0 mM dipicolinic acid for the reduction of [Fe(bpy)2(CN)2]+ and is completely suppressed with the addition of 5.0 mM EDTA (pH < 9.00), 10.0 mM EDTA (9.0 < pH 10.0), and 1.0 mM cyclam (pH > 10.0) for the reduction of [Fe(bpy)(CN)4]-. 1H NMR and UV-vis spectra show that the products of the direct (uncatalyzed) reactions are the corresponding Fe(II) complexes and, when no radical scavengers are present, L-cystine, both being formed quantitatively. The two reactions display mild kinetic inhibition by Fe(II), and the inhibition can be suppressed by the free radical scavenger PBN (N-tert-butyl--phenylnitrone). At 25 C and = 0.1 M and under conditions where inhibition by Fe(II) is insignificant, the general rate law is -d[Fe(III)]/dt = k[cysteine]tot[Fe(III)], with k = {k2Ka1[H+]2 + k3Ka1Ka2[H+] + k4Ka1Ka2Ka3{/}[H+]3 + Ka1[H+]2 + Ka1Ka2[H+] + Ka1Ka2Ka3}, where Ka1, Ka2, and Ka3 are the successive acid dissociation constants of HSCH2CH(NH3+)CO2H. For [Fe(bpy)2(CN)2]+, the kinetics over the pH range of 3-7.9 yields k2 = 3.4 ± 0.6 M-1 s-1 and k3 = (1.18 ± 0.02) × 106 M-1 s-1 (k4 is insignificant in the fitting). For [Fe(bpy)(CN)4]- over the pH range of 6.1-11.9, the rate constants are k3 = (2.13 ± 0.08) × 103 M-1 s-1 and k4 = (1.01 ± 0.06) × 104 M-1 s-1 (k2 is insignificant in the fitting). All three terms in the rate law are assigned to rate-limiting electron-transfer reactions in which various thiolate forms of cysteine are reactive. Applying Marcus theory, the self-exchange rate constant of the SCH2CH(NH2)CO2-/-SCH2CH(NH2)CO2- redox couple was obtained from the oxidation of L-cysteine by [Fe(bpy)(CN)4]-, with k11 = 4 × 105 M-1 s-1. The self-exchange rate constant of the SCH2CH(NH3+)CO2-/-SCH2CH(NH3+)CO2- redox couple was similarly obtained from the rates with both Fe(III) oxidants, a value of 6 × 106 M-1 s-1 for k11 being derived. Both self-exchange rate constants are quite large as is to be expected from the minimal rearrangement that follows conversion of a thiolate to a thiyl radical, and the somewhat lower self-exchange rate constant for the dianionic form of cysteine is ascribed to electrostatic repulsion.
Christa Jakopitsch, Holger Spalteholz, Paul G. Furtmüller, Jürgen Arnhold and Christian Obinger, Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

[Abstract]   [URL]

2008 Horseradish peroxidase, Chlorination reaction, Chlorite, Chlorine dioxide, Hypochlorous acid, Compound I, Compound II J INORG BIOCHEM, 2008, Vol 102, Issue 2, pp 293-302
Abstract: It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k1) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k2) and compound II (k3) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 × 105 M-1 s-1 (k1), 2.25 × 105 M-1 s-1 (k2), and 2.4 × 104 M-1 s-1 (k3), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 × 107 M-1 s-1 and 2.7 × 104 M-1 s-1, respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.
Christa Jakopitsch, Holger Spalteholz, Paul G. Furtmüller, Jürgen Arnhold and Christian Obinger Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

[Abstract]   [URL]

2008 Horseradish peroxidase, Chlorination reaction, Chlorite; Chlorine dioxide, Hypochlorous acid, Compound I, Compound II J INORG BIOCHEM, 2008, Vol 102, Iss 2, pp 293-302
Abstract: It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k1) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k2) and compound II (k3) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 × 105 M-1 s-1 (k1), 2.25 × 105 M-1 s-1 (k2), and 2.4 × 104 M-1 s-1 (k3), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 × 107 M-1 s-1 and 2.7 × 104 M-1 s-1, respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.
Daniel E. Otzen, Lise W. Nesgaard, Kell K. Andersen, Jonas Høeg Hansen, Gunna Christiansen, Hidekazu Doe and Pankaj Sehgal Aggregation of S6 in a quasi-native state by sub-micellar SDS

[Abstract]   [URL]

2008 Aggregation, Kinetics, SDS, Anionic surface, Unfolding BBA-PROTEINS PROTEOMICS, 2008, Vol 1784, Iss 2, pp 400-414
Abstract: Anionic surfaces promote protein fibrillation in vitro and in vivo. Monomeric SDS has also been shown to stimulate this process. We describe the dynamics of conformational changes and aggregative properties of the model protein S6 at sub-micellar SDS concentrations. S6 exhibits a rich and pH-sensitive diversity in conformational changes around 0.2–2 mM SDS, in which several transitions occur over time scales spanning milliseconds to hours. Monomeric SDS readily precipitates S6 within minutes at pH-values of 5 and below to form states able to bind the fibril-specific dye thioflavin T. At pH 5.5, the process is much slower and shows a mutagenesis-sensitive lag, leading to different forms of organized but not classically fibrillar aggregates with native-like levels of secondary structure, although the tertiary structure is significantly rearranged. The slow aggregation process may be linked to conformational changes that occur at the second-time scale in the same SDS concentration range, leading to an altered structure, possibly with unfolding around the C-terminal helix. The S6 aggregates may be differently trapped states, equivalent to pre-fibrillar structures seen at early stages in the fibrillation process for other proteins. The low quantities of anionic species required suggest that the aggregates may have parallels in vivo.
Sara Goldstein and Joseph Rabani The ferrioxalate and iodide–iodate actinometers in the UV region

[Abstract]   [URL]

2008 Ferrioxalate, Iodide–iodate, Actinometer, Quantum yield J PHOTOCHEM PHOTOBIOL A-CHEM, 2008, Vol 193, Iss 1, pp 50-55
Abstract: The ferrioxalate and iodide–iodate actinometers have been re-studied in view of apparent inconsistencies and disagreements of results obtained using different methods and laboratories. The quantum yields have been determined with the aid of highly accurate and sensitive calibrated photo-diodes in the range 205–365 nm. In the case of ferrioxalate, a pronounced change between 240 and 270 nm was observed with a plateau below 240 nm, Φ(FeII) = 1.48 ± 0.02, and above 270 nm, Φ(FeII) = 1.25 ± 0.02. The latter value agrees with other literature reports and is attributed to the known ligand to metal charge transfer band around 300 nm. A shoulder at 215–230 nm is apparently associated with the higher quantum yield below 240 nm. The quantum yield of I3- in the iodide–iodate system is essentially constant between 205 and 245 nm, Φ(I3-) = 0.92 ± 0.02. The results agree with part of the literature values and provide reliable Φ(I3-) for the range 205–290 nm for the purpose of actinometry. The steep change above 245 nm introduces a high uncertainty unless well-defined monochromatic light is used. The integrated results of both actinometers are consistent, and apparent discrepancies in literature are resolved.
Celestine N Chi, Lisa Elfstrom, Yao Shi, Tord Snall, Ake Engstrom, Per Jemth Reassessing a sparse energetic network within a single protein domain

[Abstract]   [URL]

2008 allostery, coupling energy, dynamics, energetic network of residues, PDZ domain PNAS 2008 vol. 105 no. 12 pp 4679-4684
Abstract: Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295–299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.
Dina Grohmann, Dr., Valentina Corradi, Dr., Mira Elbasyouny , Annika Baude , Florian Horenkamp, Sandra D. Laufer , Fabrizio Manetti, Dr. , Maurizio Botta, Prof. , Tobias Restle, Prof. Small Molecule Inhibitors Targeting HIV-1 Reverse Transcriptase Dimerization

[Abstract]   [URL]

2008 antiviral agents, dimerization, drug design , HIV reverse transcriptase ChemBioChem Volume 9 Issue 6, Pages 916 - 922
Abstract: The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t1/2 of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.
Hiroko Ikushiro, Shigeru Fujii, Yuka Shiraiwa, and Hideyuki Hayashi Acceleration of the substrate Cα deprotonation by an analogue of the second substrate palmitoyl-CoA in serine palmitoyltransferase

[Abstract]   [URL]

2007 palmitoyl-CoA, serine palmitoyltransferase J BIOL CHEM, 2007, Vol 282
Abstract: Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of L-serine alone to SPT leads to the formation of the external aldimine, but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a non-reactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen–deuterium exchange at Ca of L-serine is very slow in the SPT–L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the non-reactive palmitoyl-CoA facilitated a-deprotonation indicates that the a-deprotonation takes place before the Claisen-type C–C bond formation, which is consistent with the accepted mechanism of the a-oxamine synthase subfamily enzymes. Structural modeling of both the SPT–L-serine external aldimine complex and SPT–L-serine–palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP–L-serine external aldimine so that the Ca-H bond of L-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the a-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His159 with L-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.
Inna Yu. Churbanova and Irina F. Sevrioukova Redox-dependent changes in molecular properties of mitochondrial apoptosis inducing factor

[Abstract]   [URL]

2007 Apoptosis-inducing Factor, FAD, flavin, NADH, J BIOL CHEM, 2007, Vol 282
Abstract: Mitochondrial apoptosis inducing factor (AIF) is a central player in the caspase-independent cell death pathway whose normal physiological function remains unclear. Our study showed that naturally folded mouse AIF very slowly reacts with NAD(P)H (kcat of 0.2-0.01 s-1) forming tight, dimeric and air-stable FADH2-NAD(P) charge-transfer complexes ineffective in electron transfer. FAD reduction is accompanied by a conformational change involving AIF-specific N-terminal and regulatory 509-559 peptides and the active site His453, and affects susceptibility of AIF to calpain and AIF-DNA interaction, the two events critical for initiating caspase-independent apoptosis. Based on our results, we propose that formation of long-lived complexes with NAD(P)H and redox reorganization may be functionally important and enable AIF to act as a redox signaling molecule linking NAD(P)H-dependent metabolic pathways to apoptosis.
Tobias Werther, Michael Spinka, Kai Tittmann, Anja Schütz, Ralph Gobik, Carmen Mrestani-Klaus, Gerhard Hübner, and Stephan König Amino acids allosterically regulate the thiamine diphosphate-dependent α -keto acid decarboxylase from mycobacterium tuberculosis

[Abstract]   [URL]

2007 α -keto acids, Stopped-flow kinetics, Mycobacterium tuberculosis J BIOL CHEM, 2007, Vol 282
Abstract: The gene rv0853c from Mycobacterium tuberculosis strain H37Rv is coding for a thiamine diphosphate-dependent α -keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic α -keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by α -keto acids. Even more, we demonstrate that also amino acids are direct and potent activators of this ThDP-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated and implications for other thiamine diphosphate-dependent enzymes are discussed.
Shiva Bhowmik, Geoff P. Horsman, Jeffrey T. Bolin, and Lindsay D. Eltis The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation: LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION

[Abstract]   [URL]

2007 polychlorinated biphenyls, PCB, Bph, BphD, catalysis J BIOL CHEM, 2007, Vol 282
Abstract: The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 Å from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.
Nathan Cobb, Craig Hemann, Gregory A. Polsinelli, Justin P. Ridge, Alastair G. McEwan, and Russ Hille Spectroscopic and Kinetic Studies of Y114F and W116F Mutants of Me2SO Reductase from Rhodobacter capsulatus

[Abstract]   [URL]

2007 Me2SO reductase, Rhodobacter capsulatus, Dimethyl sulfoxide reductase J BIOL CHEM, 2007, Vol 282, Iss 49, pp 355199-35529
Abstract: Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me2SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me2SO to reduced enzyme, the result of lower stability of the Ered·Me2SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wild-type enzyme, consistent with Tyr-114 interacting with bound Me2SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second MoFormula O group. Reduction of the mutant with sodium dithionite and reoxidation with Me2SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This `redox-cycled` mutant exhibits a Me2SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover.
Kirill B. Gromadski, Tobias Schümmer, Anne Strømgaard, Charlotte R. Knudsen, Terri Goss Kinzy, and Marina V. Rodnina Kinetics of the Interactions between Yeast Elongation Factors 1A and 1Bα, Guanine Nucleotides, and Aminoacyl-tRNA

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2007 Yeast Elongation Factor, Guanine Nucleotides, Aminoacyl-tRNA J BIOL CHEM, 2007, Vol 282, Iss 49, pp 35629-35637
Abstract: The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Bα (eEF1Bα), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 µM, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s–1) and is accelerated by eEF1Bα by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Bα binding to eEF1A (107–108 M –1 s–1) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s–1, which is compatible with the rate of protein synthesis in the cell. eEF1A·GTP binds Phe-tRNAPhe with a Kd of 3 nM, whereas eEF1A·GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.
Gonzalo Izaguirre, Richard Swanson, Srikumar M. Raja, Alireza R. Rezaie, and Steven T. Olson Mechanism by Which Exosites Promote the Inhibition of Blood Coagulation Proteases by Heparin-activated Antithrombin

[Abstract]   [URL]

2007 Heparin , Blood Coagulation Proteases, Antithrombin, Exosites J BIOL CHEM, 2007, Vol 282, Iss 46, pp 33609-33622
Abstract: Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve the effects of these exosites on the initial Michaelis docking step and the subsequent acylation and conformational change steps of antithrombin-protease reactions, we compared the reactions of catalytically inactive S195A and active proteases with site-specific fluorophore-labeled antithrombins that allow monitoring of these reaction steps. Heparin bound to N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled antithrombins and accelerated the reactions of the labeled inhibitor with thrombin and factor Xa similar to wild type. Equilibrium binding of NBD-labeled antithrombins to S195A proteases showed that exosites generated by conformationally activating antithrombin with a heparin pentasaccharide enhanced the affinity of the serpin for S195A factor Xa minimally 100-fold. Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activator enhanced antithrombin affinity for both S195A factor Xa and thrombin at least 1000-fold. Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affinity resulted from increases in kon and decreases in koff and caused antithrombin-protease reactions to become diffusion-controlled. Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255, Glu-237, and Arg-399 made more modest contributions to these interactions; and that exosite interactions reduced koff for the Michaelis complex interaction. Together these results show that exosites generated by heparin activation of antithrombin function both to promote the formation of an initial antithrombin-protease Michaelis complex and to favor the subsequent acylation of this complex.
William C. Cooper, Yi Jin, and Trevor M. Penning Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate: THE EXAMPLE OF RAT LIVER 3α-HSD (AKR1C9)

[Abstract]   [URL]

2007 Hydroxysteroid dehydrogenases, steroid biosynthesis, enzyme kinetics J BIOL CHEM, 2007, Vol 282, Iss 46, pp 33484-33493
Abstract: Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3α-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP+ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP+ controlled kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated kcat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
Fabrice Neiers, Sanjiv Sonkaria, Alexandre Olry, Sandrine Boschi-Muller, and Guy Branlant Characterization of the Amino Acids from Neisseria meningitidis Methionine Sulfoxide Reductase B Involved in the Chemical Catalysis and Substrate Specificity of the Reductase Step

[Abstract]   [URL]

2007 methionine sulfoxide reductases (msrs), neisseria meningitidis, enzyme catalysis J BIOL CHEM, 2007, Vol 282, Iss 44, pp 32397-32405
Abstract: Methionine sulfoxide reductases (Msrs) are antioxidant repair enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The Msr family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the S and R isomers of the sulfoxide function, respectively. Both classes of Msr share a similar three-step chemical mechanism involving first a reductase step that leads to the formation of a sulfenic acid intermediate. In this study, the invariant amino acids of Neisseria meningitidis MsrB involved in the reductase step catalysis and in substrate binding have been characterized by the structure-function relationship approach. Altogether the results show the following: 1) formation of the MsrB-substrate complex leads to an activation of the catalytic Cys-117 characterized by a decreased pKapp of ~2.7 pH units; 2) the catalytic active MsrB form is the Cys-117-/His-103+ species with a pKapp of 6.6 and 8.3, respectively; 3) His-103 and to a lesser extent His-100, Asn-119, and Thr-26 (via a water molecule) participate in the stabilization of the polarized form of the sulfoxide function and of the transition state; and 4) Trp-65 is essential for the catalytic efficiency of the reductase step by optimizing the position of the substrate in the active site. A scenario for the reductase step is proposed and discussed in comparison with that of MsrA.
Yanchao Ran, Hui Zhu, Mengyao Liu, Marian Fabian, John S. Olson, Roman Aranda, IV, George N. Phillips, Jr., David M. Dooley, and Benfang Lei Bis-methionine Ligation to Heme Iron in the Streptococcal Cell Surface Protein Shp Facilitates Rapid Hemin Transfer to HtsA of the HtsABC Transporter

[Abstract]   [URL]

2007 Streptococcus pyogenes, surface protein, Shp, hemin htsA, lipoprotein J BIOL CHEM, 2007, Vol 282, Iss 43, pp 31380-31388
Abstract: The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process ~10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (ShpM66A and ShpM153A) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 µM-1, respectively, showing that the Met153–Fe bond is critical for high affinity binding and that Met66 destabilizes hemin binding to facilitate its rapid transfer. ShpM66A and ShpM153A rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4–3 s-1. Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a `plug-in` mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.
Laura M. S. Baker, Paul R. S. Baker, Franca Golin-Bisello, Francisco J. Schopfer, Mitchell Fink, Steven R. Woodcock, Bruce P. Branchaud, Rafael Radi, and Bruce A. Freeman Nitro-fatty Acid Reaction with Glutathione and Cysteine: KINETIC ANALYSIS OF THIOL ALKYLATION BY A MICHAEL ADDITION REACTION

[Abstract]   [URL]

2007 nitration, Nitro-fatty Acid, LIPIDS AND LIPOPROTEINS J BIOL CHEM, 2007, Vol 282, Iss 42, pp 31085-31093
Abstract: Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M-1s-1 and 355 M-1s-1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-{Delta}12,14-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-beta-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.