Download Authors Title Year Keywords Journal/Proceedings Emile Bol

Authors
Title
Year
Keywords
Journal/Proceeding
s
Emile Bol,
A steady-state and pre-steady-state
Nicolette J.
Broers, and
Wilfred R. Hagen
Tungsten,
J BIOL INORG CHEM,
kinetics study of the tungstoenzyme
Formaldehyde
2008, Vol 13, Iss 1, pp
formaldehyde ferredoxin
oxidoreductase,
75-84
oxidoreductase from Pyrococcus
Pyrococcus furiosus,
furiosus
Pre-steady-state
[Abstract]
2008
kinetics,
[URL]
Steady-state
kinetics
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,
Kinetic studies on the reactions of
ivadin D. Bugari
[Pd(dach)(X�Y)] complexes with
and Rudi van Eldik
some DNA constituents
[Abstract]
2008
[Pd(dach)(X�Y)] ,
J CHEM SOC DALTON
DNA, Kinetic studies
TRANS, 2008
[URL]
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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
Folding thermodynamics and kinetics
Courtemanche
and Doug Barrick
repeat protein,
PROTEIN SCI, 2008,
of the leucine-rich repeat domain of
leucine-rich repeat,
Vol 17, pp 43-53
the virulence factor Internalin B
protein folding,
[Abstract]
2008
kinetics
[URL]
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
Validation of a robust and sensitive
Freinbichler, Maria
method for detecting hydroxyl radical
Mammalia ;
A. Colivicchi,
formation together with evoked
Rodentia ; Basal
Manuela Fattori,
neurotransmitter release in brain
ganglion ; Excitatory
Chiara Ballini,
microdialysis
aminoacid ; Central
Keith F. Tipton,
[Abstract]
[URL]
2008
Vertebrata ;
nervous system ;
Wolfgang Linert
Toxin ; Rat ;
and Laura Della
Neurotoxin ;
Corte
Fluorescence ; In
vitro ; Peroxides ;
Oxygen ; Sodium ;
Microdialysis ;
Encephalon ;
Release ;
Neurotransmitter ;
J NEUROCHEM, 2008
Authors
Title
Year
Keywords
Journal/Proceeding
s
Radical ;
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,
Distinguishing Specific and
Sarah Batey,
Annette Steward,
and Jane Clarke
Multidomain
BIOPHYS J, 2008, Vol
Nonspecific Interdomain Interactions
proteins,
94, pp 622-628
in Multidomain Proteins
Interdomain
[Abstract]
2008
Interactions,
[URL]
spectrin-titin
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.;
Catalysis by the Isolated Tryptophan
Cullis, P. M.; Leys,
D.; Scrutton, N. S.
benzylamines,
BIOCHEMISTRY-USA,
Tryptophylquinone-Containing
Aromatic Amine
2008, Vol 47, Iss 1, pp
Subunit of Aromatic Amine
Dehydrogenase,
183-194
Dehydrogenase Is Distinct from Native
TTQ Mechanism
Enzyme and Synthetic Model
2008
Authors
Title
Year
Keywords
Journal/Proceeding
s
Compounds and Allows Further
Probing of TTQ Mechanism
[Abstract]
[URL]
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,
Oxygen-Induced Radical
L.-H.; Tsai, A.-L.
Intermediates in the nNOS Oxygenase
Domain Regulated by L-Arginine,
2008
nNOS Oxygenase,
BIOCHEMISTRY-USA,
nitric oxide,
2008, Vol 47, Iss 1, pp
405-420
Tetrahydrobiopterin, and Thiol
[Abstract]
[URL]
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.;
Direct Oxidation of L-Cysteine by
Stanbury, D. M.
[FeIII(bpy)2(CN)2]+ and
2008
L-Cysteine,
Inorg. Chem., 47 (3),
oxidation
1224 -1236, 2008.
[FeIII(bpy)(CN)4][Abstract]
[URL]
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
Mechanism of reaction of horseradish
Jakopitsch, Holger
Spalteholz, Paul
G. Furtm�ller,
J�rgen Arnhold
Horseradish
J INORG BIOCHEM,
peroxidase with chlorite and chlorine
peroxidase,
2008, Vol 102, Issue 2,
dioxide
Chlorination
pp 293-302
[Abstract]
[URL]
2008
reaction, Chlorite,
Chlorine dioxide,
Authors
Title
Year
Keywords
Journal/Proceeding
s
and Christian
Hypochlorous acid,
Obinger,
Compound I,
Compound II
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
Mechanism of reaction of horseradish
Jakopitsch, Holger
Spalteholz, Paul
G. Furtm�ller,
Horseradish
J INORG BIOCHEM,
peroxidase with chlorite and chlorine
peroxidase,
2008, Vol 102, Iss 2, pp
dioxide
Chlorination
293-302
[Abstract]
2008
reaction, Chlorite;
[URL]
J�rgen Arnhold
Chlorine dioxide,
and Christian
Hypochlorous acid,
Obinger
Compound I,
Compound II
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,
Aggregation of S6 in a quasi-native
Lise W. Nesgaard,
state by sub-micellar SDS
Kell K. Andersen,
Jonas H�eg
Hansen, Gunna
[Abstract]
[URL]
2008
Aggregation,
BBA-PROTEINS
Kinetics, SDS,
PROTEOMICS, 2008,
Anionic surface,
Vol 1784, Iss 2, pp
Unfolding
400-414
Authors
Title
Year
Keywords
Journal/Proceeding
s
Christiansen,
Hidekazu Doe and
Pankaj Sehgal
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
The ferrioxalate and iodide�iodate
and Joseph Rabani
actinometers in the UV region
[Abstract]
2008
[URL]
Ferrioxalate,
J PHOTOCHEM
Iodide�iodate,
PHOTOBIOL A-CHEM,
Actinometer,
2008, Vol 193, Iss 1, pp
Quantum yield
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,
Reassessing a sparse energetic
Lisa Elfstrom, Yao
network within a single protein domain
Shi, Tord Snall,
[Abstract]
[URL]
2008
allostery, coupling
PNAS 2008 vol. 105 no.
energy, dynamics,
12 pp 4679-4684
energetic network of
Ake Engstrom, Per
residues, PDZ
Jemth
domain
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Small Molecule Inhibitors Targeting
Dr., Valentina
Corradi, Dr., Mira
Elbasyouny ,
antiviral agents,
ChemBioChem Volume
HIV-1 Reverse Transcriptase
dimerization, drug
9 Issue 6, Pages 916 -
Dimerization
design , HIV reverse
922
[Abstract]
2008
transcriptase
[URL]
Annika Baude ,
Florian
Horenkamp,
Sandra D. Laufer ,
Fabrizio Manetti,
Dr. , Maurizio
Botta, Prof. ,
Tobias Restle,
Prof.
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,
Acceleration of the substrate Cα
Shigeru Fujii,
palmitoyl-CoA,
J BIOL CHEM, 2007, Vol
deprotonation by an analogue of the
serine
282
Yuka Shiraiwa,
second substrate palmitoyl-CoA in
palmitoyltransferase
and Hideyuki
serine palmitoyltransferase
Hayashi
[Abstract]
[URL]
2007
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.
Redox-dependent changes in
Churbanova and
Irina F.
Sevrioukova
Apoptosis-inducing
J BIOL CHEM, 2007, Vol
molecular properties of mitochondrial
Factor, FAD, flavin,
282
apoptosis inducing factor
NADH,
[Abstract]
2007
[URL]
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,
Amino acids allosterically regulate the
Michael Spinka,
α -keto acids,
J BIOL CHEM, 2007, Vol
thiamine diphosphate-dependent α
Stopped-flow
282
Kai Tittmann, Anja
-keto acid decarboxylase from
kinetics,
Sch�tz, Ralph
mycobacterium tuberculosis
Mycobacterium
Gobik, Carmen
Mrestani-Klaus,
Gerhard H�bner,
and Stephan
K�nig
[Abstract]
[URL]
2007
tuberculosis
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
The Molecular Basis for Inhibition of
Geoff P. Horsman,
2007
polychlorinated
J BIOL CHEM, 2007, Vol
BphD, a C-C Bond Hydrolase Involved
biphenyls, PCB,
282
Jeffrey T. Bolin,
in Polychlorinated Biphenyls
Bph, BphD, catalysis
and Lindsay D.
Degradation: LARGE
Eltis
3-SUBSTITUENTS PREVENT
TAUTOMERIZATION
[Abstract]
[URL]
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 � 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,
Spectroscopic and Kinetic Studies of
Me2SO reductase,
J BIOL CHEM, 2007, Vol
Craig Hemann,
Y114F and W116F Mutants of Me2SO
Rhodobacter
282, Iss 49, pp
Gregory A.
Reductase from Rhodobacter
capsulatus,
355199-35529
Polsinelli, Justin P.
capsulatus
Dimethyl sulfoxide
Ridge, Alastair G.
McEwan, and Russ
Hille
[Abstract]
[URL]
2007
reductase
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.
Kinetics of the Interactions between
Gromadski,
Yeast Elongation
J BIOL CHEM, 2007, Vol
Yeast Elongation Factors 1A and 1Bα,
Factor, Guanine
282, Iss 49, pp
Tobias
Guanine Nucleotides, and
Nucleotides,
35629-35637
Sch�mmer, Anne
Aminoacyl-tRNA
Aminoacyl-tRNA
Str�mgaard,
[Abstract]
2007
[URL]
Charlotte R.
Knudsen, Terri
Goss Kinzy, and
Marina V. Rodnina
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 �M, 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,
Mechanism by Which Exosites
Richard Swanson,
Heparin , Blood
J BIOL CHEM, 2007, Vol
Promote the Inhibition of Blood
Coagulation
282, Iss 46, pp
Srikumar M. Raja,
Coagulation Proteases by
Proteases,
33609-33622
Alireza R. Rezaie,
Heparin-activated Antithrombin
Antithrombin,
and Steven T.
[Abstract]
[URL]
2007
Exosites
Olson
Abstract: Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Elucidation of a Complete Kinetic
Yi Jin, and Trevor
M. Penning
2007
Hydroxysteroid
J BIOL CHEM, 2007, Vol
Mechanism for a Mammalian
dehydrogenases,
282, Iss 46, pp
Hydroxysteroid Dehydrogenase (HSD)
steroid biosynthesis,
33484-33493
and Identification of All Enzyme Forms
enzyme kinetics
on the Reaction Coordinate: THE
EXAMPLE OF RAT LIVER 3α-HSD
(AKR1C9)
[Abstract]
[URL]
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.
Authors
Title
Year
Keywords
Journal/Proceeding
s
Fabrice Neiers,
Characterization of the Amino Acids
Sanjiv Sonkaria,
methionine
J BIOL CHEM, 2007, Vol
from Neisseria meningitidis
sulfoxide reductases
282, Iss 44, pp
Alexandre Olry,
Methionine Sulfoxide Reductase B
(msrs), neisseria
32397-32405
Sandrine
Involved in the Chemical Catalysis and
meningitidis,
Boschi-Muller, and
Substrate Specificity of the Reductase
enzyme catalysis
Guy Branlant
Step
[Abstract]
2007
[URL]
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
Bis-methionine Ligation to Heme Iron
Zhu, Mengyao Liu,
Streptococcus
J BIOL CHEM, 2007, Vol
in the Streptococcal Cell Surface
pyogenes, surface
282, Iss 43, pp
Marian Fabian,
Protein Shp Facilitates Rapid Hemin
protein, Shp, hemin
31380-31388
John S. Olson,
Transfer to HtsA of the HtsABC
htsA, lipoprotein
Roman Aranda,
Transporter
IV, George N.
[Abstract]
2007
[URL]
Phillips, Jr., David
M. Dooley, and
Benfang Lei
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 �M-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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Nitro-fatty Acid Reaction with
Paul R. S. Baker,
nitration, Nitro-fatty
J BIOL CHEM, 2007, Vol
Glutathione and Cysteine: KINETIC
Acid, LIPIDS AND
282, Iss 42, pp
Franca
ANALYSIS OF THIOL ALKYLATION BY
LIPOPROTEINS
31085-31093
Golin-Bisello,
A MICHAEL ADDITION REACTION
Francisco J.
[Abstract]
2007
[URL]
Schopfer, Mitchell
Fink, Steven R.
Woodcock, Bruce
P. Branchaud,
Rafael Radi, and
Bruce A. Freeman
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.
Authors
Title
Year
Keywords
Journal/Proceeding
s
Emile Bol,
A steady-state and pre-steady-state
Nicolette J.
Broers, and
2008
Tungsten,
J BIOL INORG CHEM,
kinetics study of the tungstoenzyme
Formaldehyde
2008, Vol 13, Iss 1, pp
formaldehyde ferredoxin
oxidoreductase,
75-84
Authors
Title
Year
Keywords
Journal/Proceeding
s
Wilfred R. Hagen
oxidoreductase from Pyrococcus
Pyrococcus furiosus,
furiosus
Pre-steady-state
[Abstract]
kinetics,
[URL]
Steady-state
kinetics
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,
Kinetic studies on the reactions of
ivadin D. Bugari
[Pd(dach)(X�Y)] complexes with
and Rudi van Eldik
some DNA constituents
[Abstract]
2008
[Pd(dach)(X�Y)] ,
J CHEM SOC DALTON
DNA, Kinetic studies
TRANS, 2008
[URL]
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
Folding thermodynamics and kinetics
2008
repeat protein,
PROTEIN SCI, 2008,
Authors
Title
Year
Keywords
Journal/Proceeding
s
Courtemanche
of the leucine-rich repeat domain of
leucine-rich repeat,
and Doug Barrick
the virulence factor Internalin B
protein folding,
[Abstract]
Vol 17, pp 43-53
kinetics
[URL]
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
Validation of a robust and sensitive
Freinbichler, Maria
method for detecting hydroxyl radical
Mammalia ;
A. Colivicchi,
formation together with evoked
Rodentia ; Basal
Manuela Fattori,
neurotransmitter release in brain
ganglion ; Excitatory
Chiara Ballini,
microdialysis
aminoacid ; Central
Keith F. Tipton,
[Abstract]
[URL]
2008
Vertebrata ;
J NEUROCHEM, 2008
nervous system ;
Wolfgang Linert
Toxin ; Rat ;
and Laura Della
Neurotoxin ;
Corte
Fluorescence ; In
vitro ; Peroxides ;
Oxygen ; Sodium ;
Microdialysis ;
Encephalon ;
Release ;
Neurotransmitter ;
Radical ;
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,
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Distinguishing Specific and
Multidomain
BIOPHYS J, 2008, Vol
Sarah Batey,
Nonspecific Interdomain Interactions
proteins,
94, pp 622-628
Annette Steward,
in Multidomain Proteins
Interdomain
and Jane Clarke
[Abstract]
2008
Interactions,
[URL]
spectrin-titin
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.;
Catalysis by the Isolated Tryptophan
Cullis, P. M.; Leys,
D.; Scrutton, N. S.
2008
benzylamines,
BIOCHEMISTRY-USA,
Tryptophylquinone-Containing
Aromatic Amine
2008, Vol 47, Iss 1, pp
Subunit of Aromatic Amine
Dehydrogenase,
183-194
Dehydrogenase Is Distinct from Native
TTQ Mechanism
Enzyme and Synthetic Model
Compounds and Allows Further
Probing of TTQ Mechanism
[Abstract]
[URL]
Abstract: Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies with TTQ-dependent
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Oxygen-Induced Radical
L.-H.; Tsai, A.-L.
Intermediates in the nNOS Oxygenase
Domain Regulated by L-Arginine,
2008
nNOS Oxygenase,
BIOCHEMISTRY-USA,
nitric oxide,
2008, Vol 47, Iss 1, pp
405-420
Tetrahydrobiopterin, and Thiol
[Abstract]
[URL]
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.;
Direct Oxidation of L-Cysteine by
Stanbury, D. M.
[FeIII(bpy)2(CN)2]+ and
2008
L-Cysteine,
Inorg. Chem., 47 (3),
oxidation
1224 -1236, 2008.
[FeIII(bpy)(CN)4][Abstract]
[URL]
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
Mechanism of reaction of horseradish
Jakopitsch, Holger
Spalteholz, Paul
G. Furtm�ller,
Horseradish
J INORG BIOCHEM,
peroxidase with chlorite and chlorine
peroxidase,
2008, Vol 102, Issue 2,
dioxide
Chlorination
pp 293-302
[Abstract]
[URL]
2008
reaction, Chlorite,
J�rgen Arnhold
Chlorine dioxide,
and Christian
Hypochlorous acid,
Obinger,
Compound I,
Compound II
Abstract: It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle.
Authors
Title
Year
Keywords
Journal/Proceeding
s
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
Mechanism of reaction of horseradish
Jakopitsch, Holger
Spalteholz, Paul
G. Furtm�ller,
Horseradish
J INORG BIOCHEM,
peroxidase with chlorite and chlorine
peroxidase,
2008, Vol 102, Iss 2, pp
dioxide
Chlorination
293-302
[Abstract]
2008
reaction, Chlorite;
[URL]
J�rgen Arnhold
Chlorine dioxide,
and Christian
Hypochlorous acid,
Obinger
Compound I,
Compound II
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,
Aggregation of S6 in a quasi-native
Lise W. Nesgaard,
state by sub-micellar SDS
Kell K. Andersen,
Jonas H�eg
[Abstract]
[URL]
2008
Aggregation,
BBA-PROTEINS
Kinetics, SDS,
PROTEOMICS, 2008,
Anionic surface,
Vol 1784, Iss 2, pp
Unfolding
400-414
Hansen, Gunna
Christiansen,
Hidekazu Doe and
Pankaj Sehgal
Abstract: Anionic surfaces promote protein fibrillation in vitro and in vivo. Monomeric SDS has also been shown to stimulate
Authors
Title
Year
Keywords
Journal/Proceeding
s
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
The ferrioxalate and iodide�iodate
and Joseph Rabani
actinometers in the UV region
[Abstract]
2008
[URL]
Ferrioxalate,
J PHOTOCHEM
Iodide�iodate,
PHOTOBIOL A-CHEM,
Actinometer,
2008, Vol 193, Iss 1, pp
Quantum yield
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,
Reassessing a sparse energetic
Lisa Elfstrom, Yao
network within a single protein domain
Shi, Tord Snall,
[Abstract]
[URL]
2008
allostery, coupling
PNAS 2008 vol. 105 no.
energy, dynamics,
12 pp 4679-4684
energetic network of
Ake Engstrom, Per
residues, PDZ
Jemth
domain
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
sequence analysis is not necessarily a reporter of energetic coupling and allostery.
Dina Grohmann,
Small Molecule Inhibitors Targeting
Dr., Valentina
Corradi, Dr., Mira
Elbasyouny ,
antiviral agents,
ChemBioChem Volume
HIV-1 Reverse Transcriptase
dimerization, drug
9 Issue 6, Pages 916 -
Dimerization
design , HIV reverse
922
[Abstract]
2008
transcriptase
[URL]
Annika Baude ,
Florian
Horenkamp,
Sandra D. Laufer ,
Fabrizio Manetti,
Dr. , Maurizio
Botta, Prof. ,
Tobias Restle,
Prof.
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,
Acceleration of the substrate Cα
Shigeru Fujii,
palmitoyl-CoA,
J BIOL CHEM, 2007, Vol
deprotonation by an analogue of the
serine
282
Yuka Shiraiwa,
second substrate palmitoyl-CoA in
palmitoyltransferase
and Hideyuki
serine palmitoyltransferase
Hayashi
[Abstract]
2007
[URL]
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.
Redox-dependent changes in
Churbanova and
Irina F.
Sevrioukova
Apoptosis-inducing
J BIOL CHEM, 2007, Vol
molecular properties of mitochondrial
Factor, FAD, flavin,
282
apoptosis inducing factor
NADH,
[Abstract]
2007
[URL]
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,
Amino acids allosterically regulate the
Michael Spinka,
α -keto acids,
J BIOL CHEM, 2007, Vol
thiamine diphosphate-dependent α
Stopped-flow
282
Kai Tittmann, Anja
-keto acid decarboxylase from
kinetics,
Sch�tz, Ralph
mycobacterium tuberculosis
Mycobacterium
Gobik, Carmen
[Abstract]
[URL]
2007
tuberculosis
Mrestani-Klaus,
Gerhard H�bner,
and Stephan
K�nig
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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
The Molecular Basis for Inhibition of
Geoff P. Horsman,
2007
polychlorinated
J BIOL CHEM, 2007, Vol
BphD, a C-C Bond Hydrolase Involved
biphenyls, PCB,
282
Jeffrey T. Bolin,
in Polychlorinated Biphenyls
Bph, BphD, catalysis
and Lindsay D.
Degradation: LARGE
Eltis
3-SUBSTITUENTS PREVENT
TAUTOMERIZATION
[Abstract]
[URL]
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 � 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,
Spectroscopic and Kinetic Studies of
Craig Hemann,
Me2SO reductase,
J BIOL CHEM, 2007, Vol
Y114F and W116F Mutants of Me2SO
Rhodobacter
282, Iss 49, pp
Gregory A.
Reductase from Rhodobacter
capsulatus,
355199-35529
Polsinelli, Justin P.
capsulatus
Dimethyl sulfoxide
Ridge, Alastair G.
[Abstract]
[URL]
2007
reductase
McEwan, and Russ
Hille
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.
Authors
Title
Year
Keywords
Journal/Proceeding
s
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.
Kinetics of the Interactions between
Gromadski,
Yeast Elongation
J BIOL CHEM, 2007, Vol
Yeast Elongation Factors 1A and 1Bα,
Factor, Guanine
282, Iss 49, pp
Tobias
Guanine Nucleotides, and
Nucleotides,
35629-35637
Sch�mmer, Anne
Aminoacyl-tRNA
Aminoacyl-tRNA
Str�mgaard,
[Abstract]
2007
[URL]
Charlotte R.
Knudsen, Terri
Goss Kinzy, and
Marina V. Rodnina
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 �M, 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,
Mechanism by Which Exosites
Richard Swanson,
Heparin , Blood
J BIOL CHEM, 2007, Vol
Promote the Inhibition of Blood
Coagulation
282, Iss 46, pp
Srikumar M. Raja,
Coagulation Proteases by
Proteases,
33609-33622
Alireza R. Rezaie,
Heparin-activated Antithrombin
Antithrombin,
and Steven T.
[Abstract]
[URL]
2007
Exosites
Olson
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.
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Elucidation of a Complete Kinetic
Yi Jin, and Trevor
M. Penning
2007
Hydroxysteroid
J BIOL CHEM, 2007, Vol
Mechanism for a Mammalian
dehydrogenases,
282, Iss 46, pp
Hydroxysteroid Dehydrogenase (HSD)
steroid biosynthesis,
33484-33493
and Identification of All Enzyme Forms
enzyme kinetics
on the Reaction Coordinate: THE
EXAMPLE OF RAT LIVER 3α-HSD
(AKR1C9)
[Abstract]
[URL]
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,
Characterization of the Amino Acids
Sanjiv Sonkaria,
from Neisseria meningitidis
2007
methionine
J BIOL CHEM, 2007, Vol
sulfoxide reductases
282, Iss 44, pp
Authors
Title
Year
Keywords
Journal/Proceeding
s
Alexandre Olry,
Methionine Sulfoxide Reductase B
(msrs), neisseria
Sandrine
Involved in the Chemical Catalysis and
meningitidis,
Boschi-Muller, and
Substrate Specificity of the Reductase
enzyme catalysis
Guy Branlant
Step
[Abstract]
32397-32405
[URL]
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
Bis-methionine Ligation to Heme Iron
Zhu, Mengyao Liu,
Streptococcus
J BIOL CHEM, 2007, Vol
in the Streptococcal Cell Surface
pyogenes, surface
282, Iss 43, pp
Marian Fabian,
Protein Shp Facilitates Rapid Hemin
protein, Shp, hemin
31380-31388
John S. Olson,
Transfer to HtsA of the HtsABC
htsA, lipoprotein
Roman Aranda,
Transporter
IV, George N.
[Abstract]
2007
[URL]
Phillips, Jr., David
M. Dooley, and
Benfang Lei
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 �M-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
Authors
Title
Year
Keywords
Journal/Proceeding
s
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,
Nitro-fatty Acid Reaction with
Paul R. S. Baker,
nitration, Nitro-fatty
J BIOL CHEM, 2007, Vol
Glutathione and Cysteine: KINETIC
Acid, LIPIDS AND
282, Iss 42, pp
Franca
ANALYSIS OF THIOL ALKYLATION BY
LIPOPROTEINS
31085-31093
Golin-Bisello,
A MICHAEL ADDITION REACTION
Francisco J.
[Abstract]
2007
[URL]
Schopfer, Mitchell
Fink, Steven R.
Woodcock, Bruce
P. Branchaud,
Rafael Radi, and
Bruce A. Freeman
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.
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