Modulation of activity of NADH oxidase from Thermus thermophilusthrough change in flexibility in the enzyme active site induced by Hofmeister series anions Gabriel Zˇolda´k1, Mathias Spr
Trang 1Modulation of activity of NADH oxidase from Thermus thermophilus
through change in flexibility in the enzyme active site induced
by Hofmeister series anions
Gabriel Zˇolda´k1, Mathias Sprinzl2and Erik Sedla´k1
1
Department of Biochemistry, Faculty of Sciences, P J Sˇafa´rik University, Kosˇice, Slovakia;2Laboratorium fu¨r Biochemie, Universita¨t Bayreuth, Germany
The conformational dynamics of NADH oxidase from
Thermus thermophilus was modulated by the Hofmeister
series of anions (H2PO4,SO42–,CH3COO–,Cl–,Br–, I–,
ClO4,SCN–) in the concentration range 0–3M Both
cha-otropic and kosmcha-otropic anions,at high concentration,
inhibit the enzyme by different mechanisms Chaotropic
anions increase the apparent Michaelis constant and
decre-ase the activation barrier of the reaction Kosmotropic
ani-ons have the opposite effect Aniani-ons from the middle of the
Hofmeister series do not significantly affect the enzyme
acti-vity even at high concentration We detected no significant
changes in ellipticity of the aromatic region in the presence
of the anions studied There is a decreased Stern–Volmer
quenching constant for FAD fluorescence quenching in the
presence of kosmotropic anions and an increased quench-ing constant in the presence of chaotropic anions All of this indicates that active site flexibility is important in the function of the enzyme The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions,and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain,with potential importance in modulating extremozyme activity at room temperature
Keywords: activation; conformational dynamics; flavopro-teins; NADH oxidase; Thermus thermophilus
The native conformation of an enzyme is produced by the
complex interaction of van der Waals
interactions,hydro-gen bonds and ionic interactions These interactions
produce stability of the enzyme under physiological
condi-tions and prevent deleterious conformational changes from
perturbations in the environment that would cause
deacti-vation These interactions,however,must not result in
protein rigidity because the enzyme active site requires
flexibility for optimal catalytic function The balance of
these two tendencies is sensitively adjusted for the
physio-logical conditions at which the enzyme works Examples
of such adjustments are enzymes from hyperthermophiles
and psychrophiles which have optimal activity at high
(> 80C) and low (< 20 C) temperatures,respectively
[1,2] Enzymes from thermophiles are almost inactive at
room temperature because of polypeptide and side chain
rigidity induced by higher-order interactions within
secon-dary and tertiary structures Psychrophilic enzymes are
inactive at room temperature because the high flexibility of
their polypeptide and side chains results in partial/local or
complete unfolding of the tertiary structure Modulation of
the balance between the rigidity and flexibility of the
polypeptide and side chains can be achieved by changing the solvent properties Stabilization of psychrophilic enzymes without affecting their activity,or activation of thermophilic enzymes without affecting their stability,is interesting for both basic and applied protein chemistry
The use of chaotropic agents (urea,guanidinium hydrochloride) to activate different enzymes has been reported in several papers [3–8] The change in activity resulted from conformational changes in the tertiary and secondary structure of the enzymes studied We have shown recently that it is possible to activate NADH oxidase from Thermus thermophilus with urea without affecting the global stability of the enzyme at room temperature [8a] NADH oxidase (EC 1.6.99.3) from
T thermophilus is a dimeric flavoprotein containing one molecule of FMN or FAD per 25 kDa monomer,which catalyzes hydride transfer from NADH to an acceptor such as FAD,ferricyanide and oxygen [9] It belongs to the flavin reductase/nitroreductase family,which has similar broad substrate specificity,fold and quaternary structure [10,11] Localization of the active site of NADH oxidase at the edge of the dimeric interface (Fig 1) is in agreement with the fact that the active sites of enzymes are usually the most labile part of the enzyme structure [12] Perturbation
of either the static or dynamic state of the active site may lead to significantly changed activity Previous studies in our laboratory have indicated that activation of NADH oxidase is not achieved by conformational change but is a result of the increased dynamics of the polypeptide/side chain in the enzyme active site To substantiate these observations and analyze the role of dynamics in enzyme
Correspondence to E Sedla´k,Department of Biochemistry,Faculty
of Sciences,P J Sˇafa´rik University,Moyzesova 11,041 54 Kosˇice,
Slovakia Fax: + 421 55 622 21 24,Tel.: + 421 55 622 35 82,
E-mail: sedlak_er@saske.sk
Enzyme: NADH oxidase (EC 1.6.99.3).
(Received 26 August 2003,revised 8 October 2003,
accepted 28 October 2003)
Trang 2activity,we have investigated the effect of the Hofmeister
series of anions on the activity of NADH oxidase from
T thermophilus
The crystal structure provides information about the
flexibility of a given structure by comparison of temperature
B factors Temperature B factors are atomic mean square
displacements obtained from the intensity of the diffractive
spots [13] The absolute value of the B factor is dependent
on the refinement method and the conditions of
crystalliza-tion [14] It is therefore only correct to compare B factors
within a particular structure,although such data must also
be handled with caution Data from the crystals are
averaged over crystal space and time,therefore they reflect
crystal defects,static disorders and other parameters [15]
NADH oxidase has an overall low temperature factor for
the whole structure ( 23 A˚2) [9] that is in accordance with
the high stability of the protein conformation (Fig 1) The
flavin moiety,with a low B factor,indicates rigidity and
strong binding to the protein matrix Trp47,the only
tryptophan residue located in close proximity to the flavin
cofactor (within 10 A˚),is almost parallel to the isoalloxazine
ring,but the elevated temperature factor indicates it has
high flexibility The indole ring is stabilized through
hydrophobic interactions (side chains of Ala46,Leu49,
Phe120,Ala121,Ala122,Met123) from helix F The crystal
structure of a homologous nitroreductase in various states
shows that binding of the substrate (nicotinic acid) is
accompanied by the induced fit of helix F and helix E [10]
Rearrangement of helix F during the binding event results in
a change in the torsional angle of several residues
Remarkably,the residues that are involved in substrate
binding through changes in their dihedral angles are those
with the highest temperature factor,and are mostly from
helix F Similarly,the high B factors of helix F indicate that
it is highly flexible in NADH oxidase (Fig 1) Stabilization
or destabilization of this helix would affect interactions with Trp47 and thus the opening of the active site,which is necessary for activity (unpublished observation) This would indicate a mechanism of interaction of NADH substrate with the enzyme common to this flavoenzyme family The Hofmeister series of anions were chosen as suitable candidates for stabilization/destabilization of this part of NADH oxidase There are numerous reports on the effect
of the Hofmeister series of salts on folding and stability of proteins [16–18] and enzyme activity in both aqueous solutions [19–26] and organic solvents [27] It is generally accepted that the effect of these salts on protein results from interactions of the salt with the polypeptide chain (enthalpic contribution) and,indirectly,through effects on the water structure (entropic contribution) [28–36] For our study,we chose the Hofmeister series of anions: H2PO4,SO42–,
CH3COO–,Cl–,Br–, I–,ClO4,SCN– (ordered from kosmotropic to chaotropic) Anions are more efficient than cations in affecting the properties of polypeptide chains The anion–water interaction is stronger than the cation–water interaction,thus anions have a greater effect on water ordering The explanation for this is the asymmetry of the charge in a water molecule,with the negative end of the molecule’s dipole being nearer the center than the positive end [34,36]
The modulation of the conformational dynamics of the enzyme by the Hofmeister anions enabled us to show that both stabilization and destabilization of the active site of NADH oxidase by kosmotropic and chaotropic anions, respectively,inhibits its activity Application of the Hof-meister series of anions may be a suitable approach to modifying properties of enzymes from extremophiles The work presented is the result of a continuation of our interest
in understanding the role of flexibility for catalytic efficiency
of enzymes NADH oxidase from T thermophilus is a good candidate for such a study because the flexibility of its polypeptide chain is adjusted to the harsh conditions of thermophilic bacteria Therefore,the addition of chaotropic agents at room temperature will not significantly perturb the enzyme’s global structure [8a] but will modulate the flexibility of most of its labile parts,i.e the part of the protein structure where the active sites are usually located [9]
Experimental procedures
Analytical-grade biochemicals were obtained from Merck (Germany) Urea (high purity grade) was purchased from Sigma Urea concentrations were determined from refract-ive index measurements using an Abbe Refractometer AR3-AR6 The pH values of the solutions were measured with a Sensorex glass electrode before and after measurement at room temperature Only the measurements at which the pH change was less than 0.2 pH unit were used
Protein expression and purification The NADH oxidase from T thermophilus was overpro-duced in Escherichia coli JM 108 The purification proce-dure for the overproduced NADH oxidase was described
Fig 1 Homodimeric structure of NADH oxidase from T thermophilus
colored according to temperature B factor Low B factor (< 15 A˚2
rigid structure) is shown by a dark blue color,intermediate B factor
(30–45 A˚2) by green/yellow,and high B factor (> 60 A˚2) by red Flavin
cofactor and the closest tryptophan,Trp47,are shown The thin line
indicates dimeric interface The isoalloxasine ring of flavin is localized
in the rigid part of the homodimer,and Trp47 is localized on the most
flexible a-helix of the protein structure,helix F (shown within elliptical
traces).
Trang 3previously [37] The final product provided a single band on
a SDS/polyacrylamide gel stained with Coomassie Brilliant
Blue Before use,the protein was dialyzed against 5 mM
phosphate buffer,pH 7.0,in the absence of FAD The
specific activity of NADH oxidase is 1.9 UÆmg)1in 5 mM
phosphate buffer,pH 7.0
Steady-state kinetics
All kinetic measurements were performed on a Shimadzu
UV3000 spectrophotometer The kinetic parameters were
determined from the initial decrease in NADH absorbance
at 340 nm (e340nm¼ 6220M )1Æcm)1),at 20C
Measure-ments were performed after incubation (12 h) in 120 nM
NADH oxidase,5 mM phosphate,pH 7.0,containing
0.12 mM FAD and different concentrations of salts The
reaction was started by the addition of 180 lMNADH To
determine the Kmvalue the concentration of NADH was
varied in the range 5–200 lM It is not possible to use
NADH at higher concentrations because of its large
absorbance The data were fitted to the Michaelis equation:
m¼ Vmax
½NADH
½NADH þ KNADH
m;app
ð1Þ
where, Km, appis the apparent Michaelis constant and the
apparent Vmax is the maximum velocity for the catalytic
reaction The experimental data were also plotted according
to the Lineweaver–Burk equation and analyzed by linear
regression Similar results were obtained using both
meth-ods The apparent kcatwas determined as Vmax/[E]0,where
[E]0is the total concentration of NADH oxidase in solution
Determination of the Michaelis–Menten parameters has
not been possible in the presence of some concentrations of
iodine anions because of a spectral overlap of iodine
(product of the peroxide and iodide) and NADH At high
concentrations of rhodanide,perchlorate,sulfate and
phos-phate,the activity of NADH oxidase is very low and
determination of the Michaelis–Menten constants has large
errors
Temperature dependence of enzyme activity
Enzyme activity was determined in 5 mMphosphate buffer
containing 0.12 mM FAD and 120 nM NADH oxidase
Reactions were started by the addition of NADH to achieve
a final concentration of 0.18 mMNADH Initial velocities
were measured in the range 20–40C Temperature during
measurements was kept constant by temperature controlled
water circulation around the cuvette Temperature
depend-encies were analyzed with a simple Arrhenius equation
lnkcat¼ Ea
where, R is the gas constant (8.314 JÆK)1Æmol)1), Eais the
activation energy for the observed reaction,and C1 is a
temperature-independent constant At least five values were
plotted as ln (kcat) vs T)1and analyzed by linear regression
Coefficients of linearity were typically higher than 0.98
From comparison of the Arrhenius equation and the
transition state theory,the enthalpy (DH*) and entropy
(DS*) of activation were calculated
T kcat T
¼T
C2is the temperature-independent constant
The free energy of activation (DG*) was calculated from the equation:
Fluorescence emission spectroscopy The fluorescence steady-state measurements were per-formed on a Shimadzu RF5000 spectrofluorophotometer The fluorescence spectrum of tryptophan residues was obtained on excitation at 295 nm The cuvette contained
5 mM sodium phosphate,pH 7.0,with various concentra-tions of salts and 2.4 lMdimeric protein in a total volume of 2.5 mL Fluorescence measurements were performed at
20C Temperature was kept constant (± 0.3 C) by temperature controlled water circulation
Quenching of FAD fluorescence The fluorophores in NADH oxidase make it possible to perform fluorescence quenching experiments to investigate the dynamics of the environment near the fluorophore and the accessibility of the fluorophores to solvent Tryptophan moieties are widely used in quenching experiments NADH oxidase contains four tryptophans at different positions, which complicates a detailed analysis The flavin cofactor is another fluorophore that could be used as an intrinsic probe quenched by externally added quenchers,e.g iodide and rhodanide anions The commonly used noncharged quen-cher acrylamide is not an efficient quenquen-cher of FAD fluorescence The FAD fluorescence is not affected even at relatively high (0.2M) concentrations of acrylamide Fluor-escence quenching of the FAD was performed using iodide anions (KI) Stock solution (4M KI in 5 mM phosphate buffer,pH 7.0) was freshly prepared to avoid oxidation of iodide [38] Sodium dithionite could not be used in the stock solution of KI (inhibition of iodine formation) because of concomitant changes in the redox state of the flavin As it is
a single population of the FAD,it is possible to use a simple Stern–Volmer equation:
F0
where, KSV is the Stern–Volmer quenching constant Comparison of the values of KSVallows us to assess the accessibility of the FAD cofactor and,indirectly,the dynamics of its environment Fluorescence was monitored
at 525 nm after excitation by 450 nm in the absence (F0) and presence of various concentrations of KI (F) The linearity
of the experimental data (coefficient of linearity r 0.99) confirms the validity of the simple model (Eqn 6)
CD measurements
CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco,Tokyo,Japan) at 20C with
Trang 427.4 lM NADH oxidase in 50 mM sodium phosphate,
pH 7.0,and at different concentrations of salt A 1 cm
path-length cuvette was used for the aromatic region Each
spectrum was an accumulation of 10 consecutive scans
Results
The parameters characterizing the activity of NADH
oxidase,i.e the apparent rate constant,kcat,and the apparent
Michaelis constant, Km,strongly depend on the ionic
strength of the solution Increasing the ionic strength from
5 mM to 50 mM potassium phosphate results in a sixfold
increase in the kcatvalue,from 1.1 to 6.6 s)1,and a slight
decrease in the Kmvalue,from 8.5 to 5.2 lM In Table 1,it
can be seen that NADH oxidase is nonspecifically activated
by increased ionic strength,as all the salts studied at 0.5M
induced an increase in the kcatvalue of the enzyme However,
a further increase in ionic strength enabled us to distinguish
the effects of the different anions Anions from the middle
part of the Hofmeister series,Br–,Cl–,CH3COO–,without
significant chaotropic or kosmotropic properties did not
affect the value of kcateven at high concentrations On the
other hand,both chaotropic and kosmotropic anions caused
a decrease in the kcatvalue with increased concentration As
confirmed by parallel experiments with KCl and NaCl that
provided identical results within the margin of error,cations
do not have an effect on the kinetic parameters of NADH
oxidase Figure 2 shows the relative activity of the enzyme in
the presence of 1Mand 2Msalt concentrations Whereas the
apparent kcatdecreased in the presence of both chaotropic
and kosmotropic anions,the apparent Km significantly
increased in the presence of chaotropic anions and decreased
in the presence of kosmotropic anions (Table 1) It should be
noted that the real kcat(kcat real) is underestimated when the
substrate concentration is lower than 10· Km From the
Michaelis–Menten equation (Eqn 1),we know that in the
presence of [S]¼ 10 · Km the apparent kcatis related to
the real catalytic rate,as kcat real¼ 11/10 · kcat In the
presence of high concentrations (> 0.5M) of chaotropic salt,
the substrate (NADH) concentration [S] is related to Kmas
[S]ffi 3Km(Table 1) In this case, kcat realis related to kcatas
kcat realffi 4/3kcat However,at high concentrations of chaotropic salt,the absolute value of kcatis 7 times lower than in the presence of neutral salt Thus kcat realin chaotropic salts is related to kcat realin neutral salts as: kcat real chaotrop/
kcat real neutral¼ (4/3) · (1/7),i.e significantly less than 1 Therefore,even if Kmincreases by 2–3-fold,the bell shape
of kcat real(the relative values of kcat real chaotrop/kcat real neutral) will not be significantly affected
As the result of decreased conformational dynamics, enzymes from thermophiles have very low activity at low temperatures [39] The protein dynamics and thermal stability are inversely related to each other [40,41] The dependence of the enzyme activity on temperature in the presence of the salts was investigated to assess how the conformational dynamics of the active site is dependent on the type of salt present (Fig 2) Figure 3 shows the temperature dependence of the relative rate constant kcat
in the presence of 2Msalt For each salt, kcatat 20C was
Table 1 Apparent rate constant (k cat ),Michaelis constants (K m ) and their ratio r at various concentrations of the salts Assays were performed using
120 n M enzyme and 0.12 m M FAD in 5 m M potassium phosphate buffer and the given concentration of salts,pH 7.0 at 20 C The reaction was started by the addition of 0.18 m M NADH in the absence of salts Apparent k cat ¼ 1.10 ± 0.11 s)1, K m ¼ 8.5 ± 0.9 l M and catalytic efficiency
k cat /K m ¼ r ¼ 1.30 · 10 5
M )1 Æs)1 Errors in determination of k cat and K m are within 10% This value was calculated from several (2–5) independent measurements ND,Not determined.
Anion
k cat
(s)1)
K m (l M )
r · 10)5 ( M )1 Æs)1)
k cat (s)1)
K m (l M )
r · 10)5 ( M )1 Æs)1)
k cat (s)1)
K m (l M )
r · 10)5 ( M )1 Æs)1)
k cat (s)1)
K m (l M )
r · 10)5 ( M )1 Æs)1) SCN– 5.36 15.60 3.43 1.00 30.55 0.33 0.78 ND ND 0.49 ND ND ClO 4 5.10 15.65 3.26 0.81 44.22 0.18 0.54 ND ND 0.27 ND ND
I – 7.18 29.34 2.45 6.97 ND ND 3.86 ND ND 1.29 ND ND
Br– 7.61 20.90 3.64 6.75 21.71 3.11 6.22 22.51 2.76 5.36 28.14 1.91
Cl – 5.01 13.25 3.78 7.50 13.67 5.55 6.64 13.72 4.84 6.43 13.40 4.80
CH 3 COO – 4.82 12.86 3.75 4.82 10.05 5.80 5.36 18.94 2.83 4.95 28.12 1.76
SO42 2.68 11.25 2.40 2.19 7.23 3.03 1.07 ND ND 0.86 ND ND
H 2 PO 4 3.32 13.67 2.43 2.14 10.45 2.05 1.18 8.94 1.32 0.38 ND ND
Fig 2 Relative activity of NADH oxidase from T thermophilus in the presence of 1 M (gray histogram) and 2 M (black) sodium or potassium salts of the designated anions,in 5 m M phosphate buffer,pH 7.0,at
20 °C Activity was initiated by the addition of 0.2 m M NADH.
Trang 5taken as the reference value Figure 3 shows that the slope
of the observed dependencies increases according to the
position of the anions in the Hofmeister series,in the order
from chaotropic to kosmotropic anions This indicates that,
in the presence of chaotropic anions (SCN–,ClO4) the
activation energy is temperature independent,whereas in
the presence of kosmotropic anions (SO42–, H2PO4) it is
strongly temperature dependent
To determine how activation parameters are affected in
the presence of various concentrations of different salts,the
temperature dependencies of the rate constants were
measured at 20–40C (Supplementary material) Figure 4
shows a dependence of DG*,at 20C,on the concentration
of perchlorate,chloride and sulfate anions In the range
0.5–1.0Msalt,there is a minimum of this dependence for all anions studied Whereas in the presence of chloride (neutral) anions,the dependence achieves a local minimum,in the case of both sulfate (kosmotropic) and perchlorate (chao-tropic) anions,the observed minimum is global It should be noted that,although the observed minima are not pro-nounced,a similar tendency of DG* is observed for all anions,indicating that the observed dependencies are real
A double minimum or wide minimum,in the range 0.5– 2.0M salt,of DG* vs concentration is also observed for bromide,iodide and acetate anions,i.e anions from the middle part of the Hofmeister series The wide minimum in the case of these anions also supports the relative independ-ence of kcaton the salt concentration (Table 1) Only one minimum and one relatively sharp maximum activity of NADH oxidase is observed for both chaotropic and kosmotropic anions The DG* and kcat dependencies correlate in this sense that the minimum of DG* is located
at a similar (same) concentration range as the maximum of
kcatfor each given anion
To demonstrate that the observed changes in enzyme activity are related to conformational changes in the active site,we analyzed the CD spectra of the peptide (data not shown) and aromatic regions (Fig 5) The CD spectrum of NADH oxidase in the aromatic region consists of a positive
Fig 3 Dependence of relative activity of NADH oxidase from
T thermophilus on temperature in the presence of 2 M sodium or
potassium salts of the following anions: H 2 PO 4 (j),SO 4
2–
(~ ),Cl–(d),
Br– (.),I– (e),CH 3 COO– (h),ClO 4 (,),SCN– (r) in 5 mM
phosphate buffer,pH 7.0,at 20 °C.
Fig 4 Dependence of activation free energy (DG*) of the reaction
catalyzed by NADH oxidase from T thermophilus at 20 °C in the
presence of 2 M NaCl (d),NaClO 4 (,),or Na 2 SO 4 (~ ),in 5 m M
phosphate buffer,pH 7.0.
Fig 5 CD spectra of NADH oxidase from T thermophilus in the aromatic region in the presence of 2 M NaCl (dashed line),NaClO 4 (dotted line),or Na 2 SO 4 (thick solid line) and in the absence of salts (thin solid line),in 5 m M phosphate buffer,pH 7.0,at 20 °C Inset: Nor-malized tryptophan fluorescence (excitation wavelength 295 nm) of NADH oxidase from T thermophilus in the aromatic region in the presence of 2 M NaCl (dashed line),NaClO 4 (dotted line),ans Na 2 SO 4 (thick solid line) and in the absence of salts (thin solid line),in 5 m M
phosphate buffer,pH 7.0,at 20 C.
Trang 6band at 265 nm and negative ellipticity at 286 nm.
NADH oxidase contains four tryptophan residues in
positions 47,52,131,204 Trp131 and Trp204 are
completely exposed to the solution,whereas Trp52 is rigidly
embedded in the protein matrix Trp47 is in a sandwich-like
position toward the flavin cofactor at a distance of about
7.7 A˚ This is the only tryptophan residue suitably located
for interaction with the flavin Interestingly,this position
between Trp47 and the flavin cofactor can be achieved only
in the dimeric form of the enzyme [9] In accordance with
previously published CD spectra of the flavin oxidases [42],
the pronounced peak at 265 nm may result from an
asymmetric environment of the tightly bound flavin
cofac-tor and/or Trp47 in the active site of the enzyme,and Trp52
The small negative ellipticity at 286 nm corresponds to the
signal of tryptophan residues The CD spectrum of NADH
oxidase in the peptide region is not significantly perturbed,
even at high ionic strength (data not shown) Similarly,the
spectrum of the enzyme in the aromatic region in the
presence of 2M anions is only slightly affected A slight
decrease in the positive ellipticity at 265 nm in the
presence of perchlorate anions,i.e a decrease in the
asymmetry of the tryptophan residue and/or the flavin
cofactor in the active site,may result from dissociation of
the flavin cofactor in the presence of nucleophilic agents
[42] A 24 h dialysis of NADH oxidase in the presence of
2M perchlorate anions did not cause dissociation of the
flavin cofactor (data not shown) The CD spectrum of the
enzyme in the aromatic regions therefore probably reflects a
slight change in either the conformation or the dynamics of
the tryptophan residue in the active site
Fluorescence is the other very sensitive method of
monitoring changes in the environment close to the
fluorophores As shown in the inset of Fig 5,the presence
of 2Mchloride or 2Msulfate causes a change in
fluores-cence as compared with low ionic strength The fluoresfluores-cence
of NADH oxidase is decreased by 40% in the presence of
2Mperchlorate anions Interestingly,a similar decrease in
the fluorescence of NADH oxidase was also observed at the
concentration of urea at which activation of the enzyme
occurred (unpublished observation) A decrease in
fluores-cence further confirms that the flavin cofactor does not
dissociate from the enzyme Close localization of Trp47 and
the flavin cofactor causes resonance energy
transfer,result-ing in partial quenchtransfer,result-ing of the tryptophan fluorescence;
therefore,dissociation of the flavin would be accompanied
by an increase in tryptophan fluorescence
The results presented indicate a close relationship
between enzyme activity and the stability/conformational
flexibility of the active site The diminished enzyme activity
in the presence of a high concentration (> 1M) of
kosmotropic and chaotropic anions probably reflects high
stability/rigidity and too much flexibility of the active site,
respectively We observed that NADH oxidase from
T thermophilusat room temperature is activated 2.5-fold
in the presence of 1.0–1.5M urea This activation is
probably caused by increased conformational dynamics of
the side chains in the active site in the presence of urea If
this suggestion of a role for flexibility in enzyme activity is
correct,NADH oxidase,in the presence of kosmotropic
anions (H2PO4,SO42–) should be activated at higher urea
concentrations than in the presence of neutral and
chao-tropic anions The experiments presented in Fig 6 support this suggestion In the presence of phosphate and sulfate anions,NADH oxidase is more than 2.5 and 3.5 times more active,respectively,in the presence of urea than without urea (Fig 6) No activation,but relatively strong inhibition, was observed in the presence of the chaotropic anions, ClO4 and SCN–,as a result of increased concentrations of urea (Fig 6) No significant effect of urea (up to 2M) on the ellipticity of NADH oxidase in the aromatic region in 2M
sulfate,chloride and perchlorate anions (data not shown) further indicates that changes in NADH oxidase activity are not the result of pronounced conformational change but are probably due to changes in the dynamics of protein structure
Finally,the effect of anion-induced changes in the dynamics of the FAD microenvironment was further studied
by FAD fluorescence quenching using KI (Fig 7) The quenching of FAD fluorescence monitored at 525 nm in the presence of anions of the Hofmeister series strongly indicates
a changed flexibility of the flavin cofactor environment The effect of rhodanide,iodide and bromide anions on the dynamics of the enzyme active site was not investigated because these anions very efficiently quench FAD cence As acrylamide is a weak quencher of FAD fluores-cence,we used the efficient quenching property of iodide anions for these measurements The maximum concentra-tion of KI in quenching experiments was 0.15M,i.e the concentration of iodide anions at which no significant conformational change in NADH oxidase was observed Monitoring FAD fluorescence quenching is more advanta-geous than monitoring tryptophan fluorescence quenching because there is only one flavin cofactor and it is located in the active site of NADH oxidase The slope of the depend-encies of F0/F vs quencher in the presence of 2Mchaotropic anions is significantly higher than in the presence of 2M
neutral anions The higher quenching constant (Eqn 6) indicates an increase in the dynamics of the flavin cofactor in the presence of the chaotropic anions Analogously,a
Fig 6 Dependence of the relative activity of NADH oxidase from
T thermophilus on [urea],in the presence of 2 M NaH 2 PO 4 (j),
Na 2 SO 4 (~ ),NaCl (d),KI (e),NaClO 4 (,),or KSCN (r) in 5 m M
phosphate buffer,pH 7.0,20 °C.
Trang 7decrease in the slope of the dependencies in the presence of
kosmotropic salts,compared with neutral salts,indicates that
the active site of NADH oxidase is more rigid
Discussion
NADH oxidase from T thermophilus has,like other
enzymes from thermophiles,low activity at room
tempera-ture We have recently shown that the enzyme is activated in
the presence of a relatively low concentration ( 1M) of
chaotropic agents such as urea and guanidinium
hydro-chloride (unpublished observation) The observed activation
was not due to a conformational change but was a result of
increased conformational dynamics in the active site The
tightly bound structural water between Trp47 and the flavin
cofactor [9] was probably released in the presence of
chaotropic agents,and the active site of the enzyme opened,
facilitating the arrival of the substrate and leading to an
increased rate constant and an increased Michaelis constant
To test this suggestion,we investigated the effect of anions
of the Hofmeister series The Hofmeister series of anions
can be divided into chaotropic anions,which salt-in the
peptide groups,and kosmotropic anions,with a tendency to
salt-out nonpolar groups [32] The difference in the effect of
chaotropic and kosmotropic anions is also due to a charge
density that affects anion interactions with water molecules
[34] The combination of these effects led to the relatively
surprising bell shaped dependence of NADH oxidase activity vs 1Mand 2M anions,ordered according to the Hofmeister series (Fig 2) In fact,reports dealing with the effect of the Hofmeister series of anions on enzyme activity usually show a monotone trend,i.e enzymes are activated
by chaotropic or kosmotropic anions and inhibited by the opposite anions [21–24] Analysis of the bell shaped curve showed that the decrease in the rate constant in the presence
of chaotropic anions corresponded to an increase in the apparent Km,whereas the decrease in kcatin the presence of kosmotropic anions corresponded to the decrease in Km (Table 1) The apparent Michaelis constant measures the binding affinity of the enzyme for the substrate and can also
be used as an indirect measure of either inherent flexibility of
an enzyme molecule [43] or the conformational state of the active/binding site
The salts used,even at 2M,did not significantly affect the
CD spectrum of NADH oxidase in the peptide and aromatic regions This indicates (a) a strong interaction of the flavin cofactor with the protein matrix even in conditions that lead
to the dissociation of the cofactor from certain mesophi-lic flavin oxidases [42],and,more importantly,(b) the unchanged conformational state of the enzyme under the conditions studied The different dynamics of the enzyme active site in the presence of kosmotropic and chaotropic anions is indicated by: (a) a strong dependence of kcatvs temperature in kosmotropic anions,and a nearly independ-ent kcatvs temperature in chaotropic anions (Fig 3) and (b) positive and negative activation entropy in kosmotropic and chaotropic anions,respectively Moreover,a decrease in tryptophan fluorescence in the presence of perchlorate anions and slight changes in the CD spectra (Fig 5) indicate increased dynamics of the tryptophan residue in the active site of the enzyme,similar to results in the presence of
1.0M urea An analogous decrease in ellipticity in the aromatic region accompanied by changes in tryptophan fluorescence of the nonhomologous flavoprotein flavodoxin from Desulfovibrio vulgaris,in the presence of phosphate anions,was interpreted as an increase in the dynamics of the tryptophan residue in the vicinity of the flavin cofactor [44]
A stronger temperature dependence of kcatin the presence of kosmotropic anions indicates the presence of an energy barrier,i.e the difference between the basic and transition states On the other hand,the near independence of kcaton temperature in the presence of chaotropic anions indicates that the anions have a similar effect for temperature because the energy difference between the basic and transition states
is small This is in agreement with findings that chaotropic anions destroy the natural hydrogen-bonded network of water with effects similar to increased temperature or pressure [31],with a probable effect on the dynamics of the polypeptide/side chains of enzymes
A noteworthy observation is the linear dependence of activation enthalpy on activation entropy,a phenomenon known as entropy/enthalpy compensation,in the reaction catalyzed by NADH oxidase in the presence of salts in the concentration range 0.5–1.5M(Fig 8) It is apparent from these data that chaotropic (SCN–,ClO4, I–) and kosmo-tropic (SO42–, H2PO4) anions are localized at opposite ends
of the linear dependence,and neutral anions (Br–,Cl–,
CH3COO–) are in the middle of the dependence This also indicates that chaotropic and kosmotropic salts have
Fig 7 Dependence of FAD fluorescence in the presence of 2 M
NaH 2 PO 4 (j),Na 2 SO 4 (~ ),NaCl (d),CH 3 COONa (h),or NaClO 4
(,) on concentration of iodide anions expressed as dependence of F 0 /F vs.
concentration of iodide anions Fluorescence was monitored at 525 nm
after excitation at 450 nm in the absence (F 0 ) and presence of various
concentrations of KI (F) Steeper dependence indicates that,in the
presence of the given salt,the accessibility of the flavin cofactor or
efficiency of quenching of FAD fluorescence is higher than
depend-encies with less steep slopes The numerical value of the slope of the
dependencies is an expression of the Stern–Volmer quenching constant
(Eqn 6; coefficients of linearity for all of displayed dependencies were
r ‡ 0.99) The K SV values for the salts studied were 5.97 ± 0.26 M )1
for NaH 2 PO 4 ,5.96 ± 0.36 M )1 for Na 2 SO 4 ,14.77 ± 0.26 M )1 for
NaCl,11.76 ± 0.16 M )1 for CH 3 COONa,and 25.06 ± 0.60 M )1 for
NaClO 4 All measurements were performed in 5 m M phosphate buffer,
pH 7.0,at 20 C.
Trang 8different effects on the dynamic state of the enzyme As the
enzyme catalyzes the same reaction in the presence of
chaotropic and kosmotropic anions,the negative value of
activation entropy in chaotropic salts indicates a higher
flexibility of the basic state compared with the transition
state In other words,the difference between the activation
parameters of the enzyme in chaotropic and kosmotropic
anions is always negative as is the case of the difference in
activation parameters between psychrophilic and mesophilic
enzymes [45]
Activation of NADH oxidase by urea in the presence of
the anions studied is dependent on their position in the
Hofmeister series There is strong activation by urea in
the presence of kosmotropic anions,slight activation in the
presence of neutral anions,and deactivation in the presence
of chaotropic anions These observations indicate that the
active site of NADH oxidase is more stable in the presence
of kosmotropic anions than in the presence of chaotropic
anions The quenching experiments of the flavin cofactor
fluorescence (Fig 7) strongly support this interpretation
and strongly indicate that the anion-induced changes in the
activity of NADH oxidase are due to a change in flexibility
of the enzyme active site
These results show that anions nonspecifically activate
NADH oxidase at low concentrations (< 0.5M) This is in
accordance with the positive electrostatic potential from the
protein near the flavin cofactor which is a common feature
of homologous flavoenzymes [11] Nonspecific changes in
the exact nature of the contacts within related groups by the
anions may then change (in our case activate) the enzyme at
low (< 0.5M) concentrations of salt At higher
concentra-tions of salt,the effect of the anions is different and depends
on their position in the Hofmeister series Whereas anions
from the middle of the Hofmeister series do not affect
activity,both chaotropic and kosmotropic anions inhibit
NADH oxidase Changes in Km,Stern–Volmer quenching
constants,activation entropy and fluorescence,along with
slight changes in CD spectra,and localization of the active
site in the region with an increased temperature B factor
(Fig 1) strongly suggest that the mechanism of inhibition of chaotropic and kosmotropic anions includes a modulation
of flexibility in comparison with the optimal dynamics the active site Kosmotropic anions stabilize and increase the rigidity of the enzyme active site and thus slow the catalytic rate kcat On the other hand,chaotropic anions destabilize and increase the flexibility of the enzyme active site The increased flexibility in the substrate-binding site leads to the increase in Km,i.e a decrease in the affinity of the enzyme for the substrate The decrease in kcat, however,can be only partially explained by the observed increase in Km The main reason for a decreased kcat in the presence of chaotropic anions is probably increased dynamics in the active site which perturbs the proper position of the donor/substrate and the acceptor/flavin cofactor in the hydride transfer and/or other side chains with an active role in the catalytic site of NADH oxidase Modulation of the conformational dynamics by the Hofmeister series of anions therefore offers a simple strategy for activation of enzymes from thermophiles and psychrophiles
Proteins from thermophiles are stabilized by a combina-tion of strategies [46] An important one is the presence of optimized ionic pairs on the protein surface (electrostatic interaction),i.e where the active sites of enzymes are localized [46–48] Perturbation weakens some of the ionic interactions and may affect the mobility of the polypeptide/ side chain on the protein surface This would have a positive impact on the enzyme activity without a significant effect on the stable hydrophobic protein core
On the other hand,enzymes from psychrophiles contain a highly charged region in order to improve solvent interac-tions with a hydrophilic surface [50] Shielding of these interactions by a suitably chosen salt from the Hofmeister series,or another osmolyte,may stabilize the protein structure at increased temperature without deleterious effects on enzyme activity
Acknowledgements
We thank the Fonds of Chemischen Industrie for financial support We are also grateful for support through grants no D/01/02768 from the Deutsche Akademische Austauschdienst (DAAD),and no 1/8047/01 and 1/0432/03 from the Slovak Grant Agency We thank Norbert Grillenbeck for his technical assistance We also thank Linda Sowdal and Dr LeAnn K Robinson for their invaluable editorial help in preparing the manuscript.
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Supplementary material
The following material is available from http://blackwell publishing.com/products/journals/suppmat/EJB/EJB3900/ EJB3900sm.htm
Supplementary material S1 (A–H) Activation parameters calculated from the temperature dependencies of activity at various concentrations of salts