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Tiêu đề Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus
Tác giả Gabriel Žoldák, Róbert Šut’ák, Marián Antalı́k, Mathias Sprinzl, Erik Sedlák
Trường học P. J. Šafárik University
Chuyên ngành Biochemistry
Thể loại báo cáo khoa học
Năm xuất bản 2003
Thành phố Košice
Định dạng
Số trang 11
Dung lượng 375,48 KB

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Nội dung

The low quantum yield of tryptophan fluorescence in NADH oxidase shows efficient quenching of the trypto-phan residues in the protein.. The optimal temperature for enzyme activity at 1.25M

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Role of conformational flexibility for enzymatic activity in NADH

Gabriel Zˇolda´k1, Ro´bert Sˇut’a´k1,2, Maria´n Antalı´k1,3, Mathias Sprinzl4and Erik Sedla´k1

1

Department of Biochemistry, Faculty of Sciences P J Sˇafa´rik University, Kosˇice, Slovakia;2Department of Parasitology, Charles University, Prague, Czech Republic;3Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosˇice, Slovakia;4Laboratorium fu¨r Biochemie, Universita¨t Bayreuth, Germany

NADH oxidase from Thermus thermophilus is a homodimer

with an unknown physiological function As is typical for an

enzyme isolated from a thermophile,the catalytic rate,kcat,is

low at low temperatures and increases with temperature,

achieving an optimum at the physiological temperature of

the organism,i.e at 70 C for T thermophilus At low

temperatures,the kcatof several enzymes from thermophilic

and mesophilic organisms can be increased by chaotropic

agents The catalytic rate of NADH oxidase increases in the

presence of urea At concentrations of 1.0–1.3M urea it

reaches 250% of the activity in the absence of urea,at 20C

At higher urea concentrations the enzyme activity is

inhi-bited The urea-dependent activity changes correlate with

changes in the fluorescence intensity of Trp47,which is

located in the active site of the enzyme Both fluorescence

and circular dichroism measurements indicate that the

acti-vation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme This is not related to the global structure

of NADH oxidase The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases A comparison of the crystal structure with the activation thermodynamic parameters, DH* and TDS*,obtained from the temperature dependence

of kcat,suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase

Keywords:NADH oxidase; conformational dynamics; flavo-proteins; fluorescence quenching; Thermus thermophilus

The activity and stability of an enzyme is a compromise

between two opposing forces in the dynamics of the

polypeptide chain While the active site of an enzyme has

to have a certain flexibility to fit the incoming substrate,the

stability is related to the rigidity of the polypeptide chain

[1–3] The balance between the stability/rigidity and the

flexibility of the protein structure is achieved in the native

structure at physiological temperatures [4,5] It was

suggested nearly 50-years ago [6,7] that conformational

flexibility in the active site is important for substrate

binding,and for enzyme catalysis The highly dynamic

active site is more highly sensitive to perturbations of the

environment than the rest of the polypeptide structure,

which agrees with the observation that enzyme inactivation

precedes global unfolding of the enzyme structure [8] The

extreme stability of enzymes from thermophilic organisms

is an attractive feature for biotechnological applications [9]

On the other hand,these enzymes have low activity at

temperatures below their physiological temperature

Find-ing conditions in which an enzyme is activated but not

destabilized at low temperature is one way to increase

the catalytic efficiency of the thermophilic enzymes

Another way would be to identify the rate-limiting step

in enzyme catalysis This information may indicate a suitable amino-acid residue in the active site as a target for protein engineering that could result in activation of the enzyme [2]

Here,we report the case of a thermophilic enzyme that

is sensitive to the conformational flexibility of the active site We have studied the effect of urea on NADH oxidase (EC 1.6.99.3) from Thermus thermophilus NADH oxidase

is a dimeric flavoprotein containing one molecule of FMN

in each 25-kDa monomer,and it catalyzes hydride transfer from NADH to an acceptor such as FAD, ferricyanide,oxygen,and others [10] It belongs to the flavin reductase/nitroreductase family that has similar broad substrate specificity,similar folding and similar quaternary structure [11,12] The localization and potential physiological role of this alternative dehydrogenase in this thermophile species is not known In the course of the purification procedure,the major activity of NADH oxidase was found in the supernatant of the cell lysates The main location of the NADH oxidase activity was found in the polar aqueous solution This indicates a possible role in regulation of the cytoplasmic NADH/ NAD+moiety

The flavin cofactors,FMN and FAD,are tightly bound with dissociation constants of 10)7M )1and 10)5M )1, respectively The low temperature factor determined from the crystal structure also indicates tight binding [10] NADH oxidase is relatively rigid,however,the cofactor is located in

Correspondence to E Sedla´k,Department of Biochemistry,

Faculty of Sciences,P J Sˇafa´rik University,Moyzesova 11,

041 54 Kosˇice,Slovakia E-mail: sedlak_er@saske.sk

Enzymes: NADH oxidase (EC 1.6.99.3).

(Received 20 September 2003,accepted 22 October 2003)

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the intermonomeric interface that is a region with relatively

high dynamics [13] Binding of substrate in homologous

reductases is accompanied by the induced fit of the helix at

the intermonomeric interface The high temperature factor

of the analogous helix in NADH oxidase indicates its high

flexibility Destabilization of this helix containing Trp47

would affect the interaction of the flavin cofactor with

Trp47 in the active site of the enzyme This could be a

general mechanism of substrate/enzyme interaction in this

flavoprotein family

In the work presented here,we have shown that NADH

oxidase can be activated in low concentrations of chaotropic

agents due to increased local dynamics in the active site The

rate-limiting step in NADH oxidase is proposed to include

movement of Trp47 The observed correlation between

activity and tryptophan fluorescence can occur only when

the enzyme is in a dimeric form,indicating that NADH

oxidase is a functional homodimer

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 units were taken for further

consideration

Protein expression and purification

The NADH oxidase from T thermophilus was

overpro-duced in E coli JM 108 using recombinant plasmid

pTNADOX (ampR, tac promotor and nox gene) [14]

1 mMIPTG (Gerbu) was added after the bacterial culture

reached D600¼ 0.9–1.0 and harvested after 4–5 h The

purification procedure for the overproduced NADH

oxi-dase was described earlier [15] and used with only minor

modifications The heat treatment step was performed in the

presence of a small amount of FAD (increases the thermal

stability of the enzyme),dialyzed and loaded on a Blue

Sepharose CL-6B affinity column After the washing

procedure NADH oxidase was eluted with 1 mMNAD+

The final product was a single band on a SDS/PAGE gel

[16] stained with Coomassie Brilliant Blue Before use,the

protein was dialyzed in the absence of FAD in 50 mM

phosphate buffer,pH 7.2 The final preparation yielded

NADH oxidase with a specific activity of 11.32 unitsÆmg)1

at 20C One unit is defined as 1 lMNADH oxidized per

min

Determination of the protein concentration

The extinction coefficient (e) of the protein at 280 nm was

calculated from the number of tryptophan residues (4),

tyrosine residues (7) and cysteine residues (0) per

mono-mer using an equation in [17] The predicted molar

absorption coefficient for apoenzyme is e280¼

32 430M )1Æcm)1 The noncovalently bound cofactor

FAD also contributes to the extinction coefficient at

280 nm The molar absorption coefficient for FAD dissolved in pH 7.2 phosphate buffer,is e280¼

20 600M )1Æcm)1 Therefore,the protein concentration with the bound cofactor was determined using the extinction coefficient e280¼ 52 030M )1Æcm)1 The calcu-lated specific activity is very similar to previous data [15], provided the protein concentration was determined according to the method of Bradford

Steady-state kinetics All kinetic measurements were performed on a Shimadzu UV3000 spectrophotometer The kinetic parameters were determined from the initial decrease in the absorbance of NADH at 340 nm (e340¼ 6220M )1Æcm)1),at 20C Measurements were performed after incubation (12 h) in

120 nM NADH oxidase holoenzyme,50 mM sodium phosphate,pH 7.2,containing 0.120 mMFAD and differ-ent concdiffer-entrations of urea The reaction was started with the addition of NADH The observed rate at 340 nm is a combination of the enzyme-mediated rate changes and other rates,e.g the self-decay of NADH and the reduction

of externally added FAD The self-decay of NADH is insignificant in these conditions and needs to be taken into account only at high temperatures The externally added FAD has an absorption maximum at 375 nm,and reduction of the flavin might affect the absorbance at

340 nm To determine if the change in the redox state of exogenously added FAD contributes to the time-dependent changes in absorbance at 340 nm,related to oxidation of NADH,we have monitored the reduction/oxidation reac-tion of FAD Because it is very complicated to follow this reaction in the presence of NADH at 340 nm we have monitored the reduction/oxidation of FAD at 450 nm Our results indicated that equilibrium of the reaction has been achieved within the time ( 10 s) the instrument took to start collecting data,which is in accordance with a previously reported observation [18] Therefore,this reac-tion does not contribute to time-dependent changes in absorbance at 340 nm during measurements The oxidation rate of NADH depends on the initial flavin concentration, and saturation occurred at nearly 0.10 mM flavin In the enzyme assay the concentration of FAD was always 0.120 mM The data were fitted to the Michaelis–Menten equation where KM,app corresponds to the apparent Michaelis constant and the apparent Vmaxis the maximum velocity for the catalytic reaction The experimental data were also plotted according Lineweaver-Burk and analyzed

by linear regression Similar results were obtained using both methods

Temperature dependence of enzyme activity Enzyme activity measurements were performed in 50 mM phosphate buffer,0.120 mMFAD and 120 nMholoenzyme The reactions were started by the addition of NADH to achieve a final concentration of 0.180 mM NADH The initial velocities were measured from 20 to 40C The temperature during measurements was kept constant by temperature controlled water circulation around the cuvette Temperature dependences were analyzed with a simple Arrhenius equation

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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 Data (at least five

points) were plotted as ln(kcat) vs T)1 and 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

Tln kcat

T

¼TDS



C2is the temperature independent constant This approach

avoided any extrapolation connected with large errors in the

estimation of the activation entropy [19]

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

Using different excitation wavelengths,i.e 280,290 and

450 nm,we were able to follow changes in the environment

close to different internal chromophores,i.e Tyr,Trp and

FAD,respectively The cuvette contained 50 mM sodium

phosphate,pH 7.2,with various concentrations of urea and

2.4 lM dimeric protein in a total volume of 2.5 mL To

avoid the inner filter effect the absorbance of protein

samples was always lower than 0.1 Samples were incubated

12 h at room temperature The data from all fluorescence

and quenching experiments were collected at 25C The

quantum yields were calculated by a comparative method

using the integrated areas of fluorescence intensity for

protein samples and for free L-tryptophan [20,21] The

quantum yield of freeL-tryptophan was used as a standard

(FL-Trp¼ 0.14) [22] A similar approach was also used for

FAD in solution (FFAD¼ 0.05)

Fluorescence quenching

Quenching experiments were performed with acrylamide

(Carl Roth GmbH & Co.,Germany) A fresh 2M

acryl-amide (14.2%) solution was dissolved in 50 mM sodium

phosphate buffer,pH 7.2 Protein concentrations of

5–10 lM were used in 50 mM sodium phosphate buffer,

pH 7.2,and various concentrations of urea in a total

volume of 2.5 mL The acrylamide was added to the cuvette

in 5,10 and 20 lL aliquots After 30 s incubation the

emission spectra after excitation at 290 nm were recorded

Longer incubation times were not necessary No significant

changes occurred in the emission band even after 1 h of

incubation Therefore,a 30 s incubation interval was used

for all measurements and samples were assumed to reach

equilibrium Analysis of the experimental data was

performed using several models The Stern–Volmer

equation (Eqn 5) assumes a homogenous population of fluorophores:

F0

F ¼ 1 þ k0s0½Q ð5Þ where k0s0¼ KSVwhich is the quenching constant k0is the bimolecular quenching constant describing collisional quenching,and s0 is the fluorescence lifetime of the tryptophan residues In some cases,quenching of the tryptophan moiety could be described with a model of a single fluorophore population [23] This model was success-fully used for N-acetyl-L-tryptophanamide and also for NADH oxidase in 9Murea Equation 5 does not include static quenching,i.e the formation of a fluorophore complex with the quencher before excitation In the case

of static quenching,the dependence of F0/F on Q,as plotted, has an upward curvature due to factor e[Q]Vwhere V is the static constant [24] Data obtained from the quenching of NADH oxidase by acrylamide were impossible to fit to a simple Stern–Volmer equation due to a downward curva-ture of F0/F vs Q This is typical for heterogeneous populations of fluorophores This is not surprising because NADH oxidase contains four tryptophan residues,each with a different extent of accessibility to the quencher Quenching of the tryptophan moieties of NADH oxidase could be described in terms of accessible and nonaccessible populations using a modified Stern–Volmer equation [25]:

F0

F0 F¼

1

fa

where fais the fraction of accessible fluorophore and Kcis the effective collisional quenching constant This modified equation assumes that the population is heterogeneous and that there is a difference in the quenching behavior of the different tryptophan moieties A linear regression, F0

F 0 Fvs 1

½Q whose slope¼ 1

f a K cand intercept¼1

f awas used for data analysis Data processing was performed usingGRAFIT3.00 (Erithacus Software Ltd,Cambridge,UK)

Circular dichroism measurements

CD measurements were performed on a Jasco J-600 (Tokyo,Japan) spectropolarimeter at 20C with 29.3 lM NADH oxidase in 10 mMsodium phosphate,pH 7.2,and urea A 0.1 cm path-length cuvette was used for the peptide region and a 1 cm cuvette for the aromatic region Each spectrum was an accumulation of 4–6 consecutive scans The thermal transitions were recorded at 222 nm with a constant scan rate of 1 KÆmin)1 The temperature was measured with a PTC)348 WI Peltier block inside the cuvette The temperature calibration was performed with a Brand (Wertheim,Germany) precision thermometer

Results

Enzyme activity The catalytic mechanism of NADH oxidase is not under-stood The enzyme kinetics of NADH oxidase from

T thermophilus were analyzed using a simple Michaelis– Menten model where FAD is at a saturation level Figure 1

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shows the time-dependence of NADH oxidation monitored

at 340 nm in the absence and in the presence of 1.25Mand

4M urea Surprisingly,the activity of NADH oxidase is

increased in the presence of urea and reached its optimum at

1.25M urea The Lineweaver–Burk plot (Fig 1,inset)

indicates that the presence of low urea concentrations affects

both the apparent maximal velocity of the reaction and the

apparent Michaelis constant for NADH’s interaction with

the enzyme In the absence of urea the apparent steady-state

parameters were kcat¼ 6.6 ± 0.1 s)1 and

KM,app¼ 5.2 ± 0.2 lM,and the catalytic efficiency was

kcat/KM,app¼ 1.3 ± 0.1 · 106

M )1Æs)1 These values are similar to those published previously [15] All parameters

consist of multiple kinetic terms and could not be associated

directly with any one step in the catalytic reaction

The effect of urea was studied in detail,and the results are

shown in Fig 2 The measured parameters are summarized

in Table 1 The velocity of NADH oxidation is 2.5 fold

higher at 1.0–1.3 M urea compared to the control The

catalytic rate of NADH oxidase is also increased in the

presence of ionic chaotropic reagents such as guanidine

hydrochloride ( 0.5M) and sodium perchlorate

( 0.25M) (data not shown) In all experiments,externally

added flavin was the electron acceptor that recovered the

reduced internal flavin However,a similar activation of

NADH oxidase was also observed in the presence of

alternative acceptors such as ferricyanide (data not shown)

The presence of urea has a similar effect on both kcatand

KM,i.e the increase in kcatis associated with an increase in

KM This results in nearly constant values of kcat/KMat

different urea concentrations (Table 1) At higher

concen-trations of urea (> 2M) kcatsequentially decreases and,at

6.0Murea,the enzyme is essentially inactive

Fluorescence

NADH oxidase from T thermophilus contains many

fluoro-phore groups: seven tyrosine residues,four tryptophan

residues and the flavin cofactor per monomer The

trypto-phan residues emission spectra were followed after excitation

at 290 nm The maximum of the emission spectrum was

336 nm,i.e the maximum shifted to lower wavelengths compared to the emission spectrum of solvent exposedL -tryptophan (352 nm) (Fig 2,inset) This indicates that tryptophan residues in the NADH oxidase dimer are buried

in nonpolar regions of the protein [26] The emission band is a convoluted contribution of all tryptophan residues in the enzyme; therefore,it is difficult to determine separate quantum yields The averaged quantum yield is low (Fav¼ 0.07) The quantum yield of solvent accessible L -tryptophan is 0.14 and it increases if the -tryptophan residues are buried The low quantum yield of tryptophan fluorescence

in NADH oxidase shows efficient quenching of the trypto-phan residues in the protein Such quenching can be the result

of interactions with the flavin cofactor,the imidazole ring of histidine residues,negatively charged carboxylic groups and/

or by the highly mobile indole group of the tryptophan residues [27] Steady-state analysis of the FAD fluorescence

in NADH oxidase has shown that its emission maximum after excitation at 450 nm is centered at 522 nm This is very similar to the value of the emission maximum characteristic for free FAD in aqueous solution (emission at 525 nm) This finding is in agreement with the location of the cofactor in the crystal structure of NADH oxidase [10] The quantum yield of the flavin cofactor in NADH oxidase (F ¼ 0.02) is smaller than that of free FAD in solution (F ¼ 0.05) The position of the tryptophans and the flavin cofactor in the crystal structure of NADH oxidase is depicted in Fig 3 It should be noted that the structure shown contains FMN as the cofactor However,the exchange of FMN for FAD results in essentially an identical structure with only

Fig 1 Enzymatic oxidation of NADH by NADH oxidase from

T thermophilus monitored by absorbance at 340 nm at 0 M ,1.25 M ,and

4 M urea Changes in absorbance were normalized The curve is not

based on a theoretical analysis,it serves only to lead eyes Inset:

Line-weaver–Burk plot for NADH oxidation in the absence of urea (s) and

in the presence of 1.25 M urea (d) Assays were performed at 20 C.

Fig 2 The effect of urea concentration on the activity (d) and intrinsic fluorescence (n) of NADH oxidase from T thermophilus Values of fluorescence intensities are shown as the ratio F/F 0 ,where F 0 corres-ponds to fluorescence at 0 M urea,and similarly A/A 0 is the ratio of the activity (A) in the presence of urea and A 0 corresponds to the enzyme activity at 0 M urea Inset: Fluorescence emission spectra of NADH oxidase in the absence (solid line) and in the presence of 1.0 M urea (dashed line) Decrease in the fluorescence and the slight red-shift of the fluorescence maximum was observed at the low urea concentra-tion Activity was determined from the initial linear decrease of the absorbance at 340 nm The fluorescence measurements were per-formed with 5 l M protein using an excitation wavelength of 290 nm for tryptophan residues All experiments were performed at 20 C.

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slight conformational changes of the C-terminal end

between Glu189 and His194 to accommodate the second

phosphate group of FAD [10] Tryptophan residues in the

monomeric form of NADH oxidase are spatially separated

from the location of the flavin cofactor The distance

between N1 of the flavin cofactor and Ne1of tryptophans 47,

52,131,204 are 33.6 A˚,22.7 A˚,25.1 A˚,29.5 A˚,

respectively In the dimeric form,the distances of

trypto-phan residues 47,52,131,204 from the flavin cofactor are

7.7 A˚,16.3 A˚,12.5 A˚,25.3 A˚,respectively Interestingly,

changes in the enzyme activity correlate with changes in the

fluorescence intensity of the tryptophan residues

Fluores-cence probes the properties of the local environment of the dipole–dipole interaction rather than global structural changes in proteins As the dipole–dipole interaction decreases very steeply with distance (as 1/distance6),the relative position of Trp47 and the flavin cofactor is especially notable Moreover,the crystal structure indicates that contact between Trp47 and the cofactor is mediated through a tightly bound structural water molecule [10] This strongly indicates that the fluorescence of Trp47 is respon-sible for the observed correlation between activity and tryptophan fluorescence (Fig 2) It is not possible to exclude allosteric effects that could affect the distance between the cofactor and the other tryptophan residues This is probably not the case because we could not see significant changes in the circular dichroism spectra that would accompany such a significant conformational change (see below) In the presence of 1.0–1.3M urea,there is nearly

a 60% decrease in tryptophan fluorescence simultaneous with a slight red shift ( 5 nm) of the tryptophan emission maximum (Fig 2,inset) Tryptophan residues 131 and 204 are completely exposed to solvent while Trp52 is rigidly embedded at a distant location in the protein matrix In the case of NADH oxidase the perturbation of a microenvi-ronment,probably that of Trp47,is interrelated with the changes in activity at a narrow concentration range of urea

At higher concentrations of urea (> 7M) the fluorescence intensity sharply increases due to unfolding of the protein and dissociation of the flavin cofactor (data not shown) At

9Murea the tryptophan residues of NADH oxidase possess characteristics very similar to free L-tryptophanF  0.19 and kem¼ 350 nm The flavin fluorescence is not changed significantly in the presence of 0–7Murea (data not shown) The induced changes in enzyme activity and urea-induced protein unfolding,as monitored by fluorescence, show that inactivation of the enzyme takes place before the global unfolding of the protein

Circular dichroism The global structure of the protein may be efficiently monitored by CD spectroscopy The effect of urea on the

Table 1 Steady-state kinetic parameters (k cat and apparent K M ) at various concentrations of urea and temperature Acrylamide quenching constants and the fraction of the accessible tryptophans for NADH oxidase at various concentrations of urea Activity and quenching experiments were performed at 20 C (see Experimental procedures) Kinetic parameters were obtained by the nonlinear regression analyses of a simple Michaelis– Menten equation Quenching parameters were obtained from fitting by a modified Stern–Volmer equation (Eqn 6) Standard deviations (±) represent possible errors in the estimated parameters for straight line.

Urea ( M )

Activity

K M,app (l M ) 5.2 ± 0.2 8.8 ± 0.2 9.2 ± 0.4 13.8 ± 0.9 9.1 ± 0.4

k cat (s)1) 6.6 ± 0.1 9.9 ± 0.1 14.9 ± 0.2 15.3 ± 0.4 9.9 ± 0.2

k cat /K M,app ( M )1 Æs)1) 1.27 · 10 6

1.12 · 10 6

1.64 · 10 6

1.10 · 10 6

1.10 · 10 6

Fluorescence quenching

K c ( M )1 ) 20 ± 1 7.6 ± 0.6 6.8 ± 0.4 10.0 ± 0.1 14.9 ± 0.1

f a 0.42 ± 0.03 0.59 ± 0.17 0.71 ± 0.08 0.61 ± 0.03 0.48 ± 0.03

a

Coefficients obtained by linear regression.

Fig 3 Dimeric structure of NADH oxidase from T thermophilus.

Monomers are drawn in different greyscale All tryptophan residues

and FMN cofactors are shown Noteworthy,Trp47 is located close to

the environment of the FMN cofactor The structure was drawn using

VIEWER LITE 42 (1NOX.pdb).

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activity of NADH oxidase was therefore investigated by

circular dichroism at various concentrations of urea

(Fig 4) The shape of the spectra in the far UV region is

typical for a mixture of a-helix and b-sheet elements in

the secondary structure No apparent differences were

observed in ellipticity in the peptide region in the absence

or in the presence of 6.7Murea The secondary structure

of the enzyme is unaffected even at high concentrations of

urea indicating an extreme resistance to urea-induced

perturbations The ellipticity in the near UV region is

characteristic for aromatic residues – tryptophan,tyrosine,

and the flavin cofactor Contrary to the situation in the

peptide region,the aromatic region is sensitive to urea

(Fig 4B) The proximity of Trp47 to the flavin cofactor

induces asymmetry in the tryptophan environment that is

likely to result in a strong positive signal in the aromatic

region The addition of urea causes gradual changes in the

near UV spectrum (Fig 4B,inset) accompanied by a

decrease in ellipticity at 265 nm and a slight shift to longer

wavelengths The isodichroic point at about 270 nm

indicates that the conformational transition has a

two-state character As observed by fluorescence measurements,

the circular dichroism results confirm that the inactivation

of the enzyme at high concentrations of urea (> 6M) is

not accompanied by the global unfolding reaction The

gradual changes in ellipticity in the aromatic region

indicate local conformational changes and/or changes in

the tertiary structural dynamics in the environment of the

flavin cofactor

Thermal stability of the active site and global structure

of the enzyme

The temperature dependence of the enzyme activity was

measured as an indicator of stability of the active site

Unfolding of secondary structure is related to global unfolding of protein structure The ellipticity at 220 nm was therefore measured to assess the stability of the global structure (Fig 5) In the absence of urea,the enzyme achieves its maximal activity at 70 C which is close to the optimal temperature of T thermophilus [15] In the condi-tions where the maximal activity of the enzyme at room temperature was achieved,i.e in the presence of 1.25M urea,the stability of the active site of the enzyme is significantly perturbed The optimal temperature for enzyme activity at 1.25M urea was shifted by )25 C from the optimal temperature of NADH oxidase in the absence of urea (Fig 5) An additional increase in the urea concentration (> 2.0M) had no significant effect on the optimal temperature of the enzyme but reduced the maximal enzyme activity (Fig 5,Table 1) The transition temperature, Ttrs,of unfolding of the secondary structure,is represented by the position of the peak maximum of the first derivative of ellipticity at 220 nm dQ/dT Global stability, characterized by this transition temperature,is significantly higher than the thermal stability of the active site of the enzyme (Fig 5) In the absence of urea, Ttrs¼ 88.6 C, about 15C higher than the temperature of the physiolo-gical milieu of T thermophilus In the presence of low concentrations of urea,i.e 2.5M,the transition temperature decreases only by about 3C Even in the conditions where the enzyme is completely inactive,i.e at 6.7M urea,the thermal transition of the protein secondary structure has a sigmoidal shape with Ttrs¼ 71.6 C (data not shown) In summary,the active site of the enzyme is considerably more sensitive to temperature-induced perturbation than the global structure of NADH oxidase This is most pro-nounced at low concentrations of denaturant where the optimal activity of the enzyme is achieved at the expense of the flexibility/stability of the active site of the enzyme

Fig 4 Circular dichroism spectra of NADH oxidase from T

thermo-philus in the peptide (A) and aromatic (B) regions in the absence and

presence of urea (A) 0 M urea (solid line),6.7 M urea (dashed-double

dotted line) (B) 0 M urea (solid line),0.9 M (dashed line),1.7 M (dotted

line),3.5 M (dash-dotted line),and 6.7 M urea (dash-double dotted

line) Measurements were performed on a Jasco J-600

spectropola-rimeter with 29.3 l M NADH oxidase A 0.1 cm path-length cuvette

was used for far UV and a 1 cm cuvette for the aromatic region Inset:

dependence of difference in the CD spectra at 250–320 nm on

con-centration of urea The curve in the inset is not based on a theoretical

analysis,it serves only to lead the eyes.

Fig 5 Normalized activity and the first derivative of the melting curve

of NADH oxidase monitored by ellipticity at 220 nm Activity was measured in 50 m M phosphate buffer,pH 7.2,in the absence of urea (d),1.25 M urea (n) and 2.0 M urea (h),respectively Heat denatur-ation was measured in 10 m M phosphate buffer,pH 7.2,in 0 M urea (solid line),1.3 M urea (dotted line) and at 2.6 M urea (dashed line) The lines are not based on a theoretical curve but only serve to lead the eyes Transition temperature at 0 M urea T trs ¼ 88.6 C,1.3 M urea

T trs ¼ 88.4 C and at 2.6 M urea T trs ¼ 85.6 C.

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To address quantitatively the activation of NADH

oxidase at low concentrations of denaturant,the activation

parameters DH* and DS* of the reaction were determined

(Fig 6) The activation parameters were calculated

accord-ing equations 1–3 The urea effect on DH* and TDS*

indicates that both parameters reach a minimum at the urea

concentration at which enhancement of activity is observed

(Fig 2) The Arrhenius plot in the absence of urea is linear

from 20 to 65C without any apparent curvature or break

(Fig 6,inset) as was reported for some other enzymes from

thermophiles [28] Interestingly,the Arrhenius plots in the

absence and in the presence of 1.0 M urea intersect at

 60 C This indicates that the activity of NADH oxidase

is the same in both conditions at this temperature

Consequently,the enzyme was not activated by the addition

of urea at 60C (data not shown)

Fluorescence quenching

In an effort to monitor the accessibility of the tryptophan

residues as an indicator of the dynamics of the active site,

quenching experiments were performed Acrylamide was

used to quench the fluorescence of the tryptophan residues

It is known to be an effective quencher of the fluorescence of

tryptophan residues that are completely or partially exposed

to solvent [29] Quenching experiments enabled us to

determine the effective quenching constants Kc,and the

number of accessible tryptophan residues fa NADH

oxidase contains four tryptophan residues in different

locations within the enzyme that prevents a detailed

analysis A comparison of the parameters determined by

quenching,however,enabled us to extract information about changes in the accessibility of the tryptophan residues

in NADH oxidase (Table 1) Quenching of N-acetyltrypto-phanamide,as a model compound that is fully accessible to the solvent,is characterized by Kc¼ 35.7 ± 0.3M )1and

fa¼ 1.02 ± 0.01 (Eqn 6) If the simple Stern–Volmer model is used we obtained KSV¼ 37 ± 2M )1(Eqn 5) In the absence of urea,the effective quenching constant for NADH oxidase was Kc¼ 20 ± 1M )1and the fraction of accessible fluorophore fa¼ 0.42 ± 0.03,indicating that

 2 tryptophan residues are accessible to solvent (Fig 7) which agrees with the crystal structure that shows that Trp131 and Trp204 are completely exposed to the solvent [10] Iodide anions are another type of quencher that,due to the negative charges,is only accessible to protein surfaces The fraction of fluorophores accessible to iodide anions was

fa¼ 0.41 ± 0.02 The activity and the conformation of the active site of NADH oxidase are very sensitive to both the ionic strength and the type of anions (G Zˇolda´k,M Sprinzl and E Sedla´k,unpublished results) Therefore,for further quenching experiments we have used only uncharged acrylamide Table 1 shows that the fraction of exposed tryptophan residues of NADH oxidase increased in 1.0M urea to the value fa¼ 0.71 ± 0.08 This value corresponds

to the exposure of three tryptophan residues to solvent To

be certain that acrylamide does not affect the conformation

of the enzyme active site,we have determined the enzyme activity in the presence of up to 180 mMacrylamide At such relatively high concentrations of acrylamide the enzyme activity was only slightly diminished, 10–15%,compared

to the sample without acrylamide This indicates that acrylamide does not significantly affect the conformation of the enzyme active site This agrees with previously published results that acrylamide does not seriously perturb the native

Fig 6 Activation parameters DH* (d) and TDS* (s) as a function of

urea concentration Values were obtained from kinetic experiments

described in Experimental procedures using Eqns 1–3 Arrhenius plots

were analyzed at various concentrations of urea and temperatures in

the range from 20 to 40 C Errors were calculated according to the

deviation from linearity of the Arrhenius plots The lines are not based

on a theoretical curve,but only serve to lead the eyes Inset: Arrhenius

plots of NADH oxidase in the absence and in the presence of 1.0 M

urea,respectively The plots intersect at  60 C,i.e the activity is

equal at this temperature,indicating that the dynamics of the active site

are comparable.

Fig 7 Modified Stern–Volmer plots (Eqn 6) for acrylamide quenching

of tryptophan fluorescence at 0 M urea (d) and 1.0 M urea (s) Lines were obtained from the linear regression analysis F 0 , F is fluorescence

in the absence and presence of acrylamide, DF represents the difference

F 0 –F For 0 M urea: y ¼ 0.121Æx + 2.41 (r ¼ 0.990) For 1.0 M urea:

y ¼ 0.210Æx + 1.40 (r ¼ 0.990) For comparison,the acrylamide quenching of N-acetyl- L -tryptophanamide (NATA) fluorescence is shown (dashed line) NATA is a tryptophan analogue completely exposed to molecules of solvent and the quencher.

Trang 8

conformation of proteins because the enzyme activity of a

number of proteins is unaffected in the presence of

acrylamide [29]

Discussion

Activation of NADH oxidase is caused by an increase in

the conformational dynamics of the enzyme active site

NADH oxidase from T thermophilus,has diminished

activity at low temperatures,similar to many enzymes from

thermophilic organisms [4,30] The activation of NADH

oxidase at low concentrations of chaotropic agents may

result from: (a) a conformational change and/or (b)

increased dynamics in the enzyme active site or (c)

destabilization of the enzyme-product complex Several

types of electron-acceptors used (FMN,FAD,oxygen,

ferricyanide) gave similar results This indicates that

desta-bilization of the enzyme-product complex is not the

rate-limiting step in NADH oxidase catalysis It also indirectly

indicates that the kinetic mechanism of NADH oxidase is

the ping-pong reaction found in other homologue oxidases

[31] The activation is due to conformational and/or

dynamic changes in the enzyme active site This agrees with

previously published observations that activation of

differ-ent enzymes at low concdiffer-entrations of chaotropic agdiffer-ents was

associated with conformational changes in the tertiary

[32–37],and secondary [38] structure of the enzymes,or the

dynamics of the enzyme active site [39,40]

NADH oxidase activation correlates with changes in

tryptophan fluorescence Although the enzyme contains 4

tryptophan residues,only one tryptophan,Trp47,has a

suitable spatial location The distance and the orientation of the dipole moment (1La) towards the isoalloxazine ring of the flavin cofactor enables us to monitor static or dynamic conformational changes in the active site (Figs 3 and 8) It should be emphasized that the proper position of a tryptophan residue towards the flavin cofactor is possible only in the dimeric structure of NADH oxidase None of the tryptophan residues is close to the binding site of the flavin cofactor in the monomeric form of the enzyme (Fig 3) The correlation of the decrease of tryptophan fluorescence and enzyme activity at low concentrations of urea strongly indicates that NADH oxidase forms functional dimers Although the dimeric structure is obvious from the crystal structure [10],it was not apparent from the properties of the enzyme in solution [15]

The enzyme concentration (80-fold difference) had no effect on the urea–induced activity changes This implies that the quaternary structure of NADH oxidase is intact in the range of urea concentrations at which enzyme activation occurred The effect of urea on the level of the tertiary but not the secondary structure of the enzyme active site is indicated by: (a) a significant shift of the optimal temperature/activity profile to lower values and unaffected thermal stability of global folding (Fig 5),(b) ellipticity changes in the aromatic region (Fig 4B) and unperturbed ellipticity in the peptide region (Fig 4A),and (c) a change in intensity and a slight shift in the maximum position of tryptophan fluorescence without any other apparent conformational changes There is a well-known relationship between protein stability and protein flexibility/rigidity that assumes the rigidity of the polypeptide chain is a prerequisite for global protein stability This relationship is supported both experimentally [1,4,41,42] and theoretically [3] On the other hand,an inverse relationship exists between rigidity and enzymatic catalysis [43,44] Urea-induced perturbation of the environment of the active site of NADH oxidase results

in a partial exposure of buried Trp47 to solvent due to the increased dynamics of the active site This is supported by (a) the slight shift in the maximum of tryptophan fluores-cence to higher wavelengths (Fig 2),(b) insignificant changes in the ellipticity in the aromatic region,and, importantly (c) an increased fraction of tryptophan residues made accessible by the addition of a quencher such as acrylamide (Fig 7) The higher dynamics of the active site is also indirectly reflected by (a) a)25 C shift of the optimal temperature and (b) an increase in the apparent KMvalues for NADH binding to the enzyme (Table 1) The pro-nounced local destabilization of the active site and the unaffected global stability (Fig 5) agrees with observations that the active site is the most flexible and thus the most labile part of the enzyme [2] Therefore,the enzyme active site undergoes inactivation at milder conditions than is necessary for global unfolding [8]

Protein dynamics are characterized by motion on a broad spectrum of time scales Not all time scales are relevant or significant for the stability or activity of an enzyme [45] Because the lifetime of the excitation state of tryptophan is several nanoseconds [24],the activation of NADH oxidase must occur on a nanosecond time scale This matches the observed rate constants for the process of stacking and unstacking bases in nucleic acids that is in the range of

106)107s)1[46,47]

Fig 8 Localization of Trp47,Wat42 and FMN in the dimeric interface

of NADH oxidase from T thermophilus The individual monomers are

designated by the letters A and B The side chain of Trp47 is almost

parallel to the flavin system The water molecule forms hydrogen

bonds to the backbone nitrogen of Trp47 and to O2 of the ribityl chain

and is located in the middle of the flavin system with distances of 3.3 A˚

to N10 and 3.7 A˚ to N5 Arrows indicate the possible flipping

move-ment of the Trp47 side chain induced by low concentrations of urea

(1.0–1.5 m) or by incoming substrate – the nicotinamide ring The

structure was drawn using VIEWER LITE 42.

Trang 9

The determination of the activation parameters shows (a)

positive values of DH* and TDS* in the absence of urea and

(b) a decrease of both parameters by 15–20 kJÆmol)1in

the range of urea concentrations where the enzyme

activa-tion occurs (Fig 6) Because urea effects both of the

activation parameters,the so-called enthalpy/entropy

com-pensation [19,48,49], tells us that DH* and TDS* have

opposite effects on the activity of the enzyme The decrease

in DH* diminishes the energy barrier of the reaction,

whereas the decrease in DS* decelerates the catalytic action

The decrease in both parameters indicates that the ground

and transition states are similar The positive entropy seems

surprising,at first glance,for a process that involves a tight

interaction of NADH and FAD in the active site of the

enzyme This is probably due to the structural role of the

water molecule in the enzyme active site [50] The observed

decrease in both activation parameters at  1.5M urea

(Fig 6) indicates that the difference between the ground and

the transition state was reduced The effect of a low urea

concentration (1.25M) might be interpreted as a breakage

of a noncovalent bond in the active site that reduces its

rigidity resulting in the negative affects on the activity of the

enzyme at low temperature The value of the decrease in

DH* 15–20 kJÆmol)1corresponds to the loss of about one

hydrogen bond and the location of the structural water

molecule in the active site The crystal structure of the

enzyme supports the inclusion of the structural water

molecule in catalysis

Implications of the observed changes for enzyme

catalysis and conformational changes

Generally speaking,proteins have more mobility in the

side chains than in the peptide backbone This is

especially true for side chains that participate in

enzy-matic catalysis [51] Analysis of the cofactor in the

binding site from the crystal structure shows an

inter-action between the flavin cofactor,the water molecule

and Trp47 (Fig 8) [10] The side chain of Trp47 is almost

parallel to the isoalloxazine ring; however,the elevated

temperature factor and the weaker electron density

indicate it is flexible The 6–7 A˚ gap between this side

chain and the flavin ring contains some diffused electron

density and one well defined and tightly bound water

molecule This water molecule forms hydrogen bonds

with the backbone nitrogen of Trp47 and the O2 of the

ribityl chain,and it is located in the middle of the flavin

system with distances of 3–4 A˚ to the flavin nitrogens

[10]

NADH oxidase and three homologous flavoproteins

form a novel flavoprotein family in which all members

contain an aromatic amino-acid residue (Phe,Trp) that

interacts with the isoalloxazine ring of the flavin cofactor

[31] This conserved interaction may play an important

role in the catalytic mechanism of the flavoproteins In

fact,the crystal structure of the nonhomologous enzyme

NADPH-cytochrome P450 oxidoreductase,with NADP+

shows nicotinamide access to FAD is blocked by a

tryptophan residue that stacks against the isoalloxazine in

the flavin ring [52] It has been proposed that the

tryptophan residue acts as a switch – when reduced

substrate,NADPH,enters the active site an interaction

between the isoalloxazine and nicotinamide rings is able

to displace the tryptophan residue After the substrate is oxidized to NADP+the interaction between the nicotin-amide ring and the flavin cofactor weakens and the indole ring of the tryptophan displaces the oxidized substrate from the binding site This mechanism may be common

in the catalytic actions of flavoproteins A similar movement of aromatic amino-acid residues in active sites

of enzymes after interaction with substrate has been proposed for other flavoproteins [53]

In NADH oxidase,the binding site of the incoming substrate (NADH) is blocked by a water molecule tightly bound to the flavin cofactor and Trp47 Thus,the nicotin-amide ring has to displace the water molecule to achieve the proper position for hydride transfer to the cofactor Breaking of the hydrogen bond(s) which displaces the water molecule,and the concomitant local conformational change

in the enzyme active site might be the rate-limiting step in NADH oxidation We speculate that this is the mechanism

by which urea at low concentrations activates NADH oxidase at room temperature It perturbs hydrogen bond(s)

in the active site between the flavin cofactor,the water molecule and Trp47 (decrease in DH* by  15–20 kJÆ mol)1) This leads to release of strain in the active site and an increase in the dynamics of the Trp47 side chain (decrease in the tryptophan fluorescence and a slight red shift of the fluorescence maximum),weakens the water molecule’s interaction with the flavin cofactor and opens the active site (increased fa value from quenching experiment) The isodichroic point in the aromatic region of the circular dichroism spectrum (Fig 4B) indicates a two state character for the conformational change The flavin-aromatic amino acids probably move from closed (buried and rigid) to open (exposed and dynamic) in the active site In fact,it has been shown that the equilibrium between solvent-exposed and

buried forms of the flavin cofactor may be important in the catalytic mechanism of flavoproteins [54] Although our results strongly suggest that Trp47 has a role in enzymatic catalysis,they are not conclusive The current work; however,identifies Trp47 as a good candidate for site directed mutagenesis to elucidate the rate-limiting step in the NADH oxidase catalysis

The increased dynamics due to urea-induced perturbation

of hydrogen bonds decreases the energy needed to go from the ground state to the transition state in the active site of NADH oxidase from T thermophilus at room temperature The changes in the dynamics of the active site of NADH oxidase at room temperature caused by changes in solvent properties,pH,and the presence of chaotropic anions further indicate an important role for dynamics/plasticity in the enzyme catalysis

Acknowledgements

The authors would like to thank the Fonds der Chemischen Industrie for financial support We are also grateful for support through grants

No D/01/02768 from the Deutsche Akademische Austauschdienst (DAAD),no 1/8047/01 and 1/0432/03 from the Slovak Grant Agency, and an internal grant from the UPJS Faculty of Sciences (VVGS 2002) for E.S and G.Zˇ We thank Norbert Grillenbeck for his technical assistance The authors wish to thank Linda Sowdal for her invaluable editorial help in preparing the manuscript.

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