210 Biochimica et Biophysica Acta 913 (1987) 210-218 Elsevier

BBA 32860

A fluorescence and NMR relaxation study of thermally-induced conformational changes in liver alcohol dehydrogenase

Zygmunt Wasylewski a, Piotr Sucharski a, Adam Wolak b and Maurice R. Eftink c

,7 Institute of Molecular Biology, Department of Biochemistry and b Institute of Physics, Jagiellonian University, Krakbw (Poland) and c Department of Chemist~, The University of Mississippi, University, MS (U.S.A.)

(Received 30 January 1987)

Key words: NMR relaxation; Alcohol dehydrogenase; Fluororescence; Protein conformation; Thermal transition; (Horse liver)

Fluorescence and NMR relaxation studies have been performed on horse liver alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of a noncovalently bound apolar probe, 2-(p- toluidinyl)naphthalene-6-sulfonic acid (TNS), indicate that a significant thermal transition occurs in the protein in the range of temperature 0-40 o C, and that there are different temperature-dependent forms of the enzyme. The transition between these forms is affected by the binding of specific ligands to the enzyme's active site. Time-resolved fluorescence studies of the two tryptophan residues in the enzyme suggest that this thermal transition occurs around tryptophan-314, which is buried near the intersubunit region. Binding of nucleotide to the enzyme causes a decrease in spin-lattice relaxation time, T~, which may result from a decrease in the number of water molecules bound to the protein. The observed results may be due to the interactions between the structural domains into which the monomer of the protein is folded.

Introduction

Alcohol dehydrogenase from horse liver is one of the best studied oligomeric enzymes. The en- zyme is a dimer of 40 kDa per subunit. Each subunit consists of a catalytic domain and a nucleotide-binding domain [1]. X-ray structure analysis of the crystalline state indicates that, in the presence of coenzyme, liver alcohol dehydro- genase undergoes a large conformational change

Abbreviations: TFE, trifluoroethanol; TNS, 2-(p-toluidinyl)- napthalene-6-sulfonic acid.

Correspondence: Z. Wasylewski, Institute of Molecular Bi- ology, Department of Biochemistry, Jagiellonian University, Krak6w, Poland.

from an open to a closed form [2-5]. The gross conformational changes can be described as a rotation of the catalytic domains with respect to the central core of the molecule, together with a narrowing of the coenzyme-binding cleft [5].

Tryptophan fluorescence has been widely used as a sensitive probe for monitoring conforma- tional changes in proteins. Liver alcohol dehydro- genase is an attractive protein for fluorescence studies, since each of the enzyme's subunits pos- sesses only two tryptophan residues. X-ray crystal- lographic studies show that Trp-15 lies near the surface and Trp-314 is extensively buried near the subunit's interface region [2].

It is known that the temperature dependence of fluorescence properties of proteins, in the nonde- naturing range of temperature, is determined by

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

the thermal dependence of the dynamic mobiliby of the protein structure surrounding the fluoro- phore [6,7]. The changes in thermal fluorescence quenching curves, under varying conditions of ligands, should express the conformational flexi- bility of enzymes containing a bilobed structure [8,9]. In fact, thermal transitions which depend on substrate binding are found in thiosulfate sulfur- transferase [8], and hexokinase [9]. These enzymes, just like a monomer of alcohol dehydrogenase, consist of a single polypeptide that is folded into two lobes and separated by a deep cleft, into which substrates are bound [1].

It is also known that the nuclear magnetic relaxation of aqueous protein solutions is a sensi- tive means for studying changes in protein struc- ture [10,11]. Proton spin relaxation time, T 1, mea- surements can give information about water mole- cules that are tightly bound on the surface of protein molecules in solution.

In the present study of liver alcohol dehydro- genase, the temperature dependence of the fluo- rescence of its tryptophan residues, as well as of a noncovalent ly bound apolar probe, 2-(p- toluidiny)napthalene-6-sulfonic acid, are mea- sured under varying conditions of ligand binding. Also the nuclear spin-lattice relaxation time, T 1, of bound water is measured. These measurements report the presence of a significant thermal transi- tion in the protein in the nondenaturing range of temperature.

Materials and Methods

For steady-state fluorescence and NMR mea- surements, alcohol dehydrogenase was prepared from fresh horse liver as described by Dalziel [12]. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed only one elec- trophoretic band. This indicates high purity of the enzyme. For fluorescence lifetime measurement the enzyme was purchased from Calibochem as a crystalline suspension. The protein concentration was determined from absorbance measurements using E~8~0 equal to 4.6 [13]. The enzyme's activity was measured by following the absorbance change at 340 nm as NAD + was converted to NADH, with ethanol as a substrate.

The coenzyme NAD + was obtained from

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Sigma Chemical Co., trifluoroethanol was pur- chased from Aldrich Chemical Co., L-tryptophan was from Reanal, Hungary, and 2-(p-toluidi- nyl)napthalene-6-sulfonic acid was from Molecu- lar Probes (Piano, TX, U.S.A.). All other reagents were analytical grade.

All measurements were performed in 0.05 M phosphate buffer (pH 7.5). Before each experi- ment the enzyme was exhaustively dialyzed for about 15 h against phosphate buffer at 4°C. Material which remained undissolved at the end of dialysis was removed by centrifugation or by ultrafiltration through a Millipore filter.

Steady-state fuorescence measurements were recorded with a laboratory-made photon-counting spectrofluorometer described previously [14]. In- trinsic fluorescence quantum yields were evaluated by comparing the area under the fluorescence

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60

3.0

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40 20 I I I

?

0

I L I I I I 3.2 3.4 3.6

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Fig. 1. Plot of the effect of temperature on the intrinsic fluorescence quan tum yiled of liver alcohol dehydrogenase excited at 295 nm in 0.05 M phosphate buffer (pH 7.5). O, alcohol dehydrogenase alone; O, alcohol dehydrogenase binary complex in the presence of 0.5 mM NAD+; ( + ) alcohol dehydrogenase ternary complex in the presence of 0.5 mM N A D + and 10 m M TFE. Protein concentration was 0.5 mg /ml .

212

spectrum of the protein sample with that of an aqueous t ryptophan solution as a reference (quantum yield equal to 0.14 [15]). Fluorescence was excited at 295 nm with a monochromator bandwidth set at 3.5 nm. Due to the comparative character of the fluorescence quantum-yield mea- surements in the presence of 0.5 mM N A D +, the fluorescence spectra of the protein solution have not been corrected according to the attenuation of the excitation beam by the presence of the ligand. This inner filter effect leads to about 25% lower values of the quantum yield in the case of the binary and ternary complexes. However, it should be pointed out that the slope of the Arrhenius plots presented in Fig. 1, as well as the calculated values of activation energies, would not be altered. The fluorescence spectra of alcohol dehydrogenase containing TNS at a concentration of 63/~M were obtained with an excitation wavelength of 366 nm and were recorded from 370 to 470 nm. In each fluorescence measurement the enzyme concentra- tion was 0.5 m g / m l and the concentration of both trifluoroethanol and ethanol were equal to 10 mM. The concentration of N A D + was 0.5 raM. The cuvette temperature was controlled by a circula- ting water, to an accuracy of + 0.2 o C.

Fluorescence lifetimes were measured with an ISS variable-frequency phase and modulation flu- orometer. The excitation source was a 150 W xenon lamp, and fluorescence phase shift and modulation data were measured at modulation frequencies ranging from about 5 to 200 MHz, using p-terphenyl in ethanol as a reference fluoro- phore (T = 1.0 ns). The sample was excited using an interference filter centered at 289 nm (half bandpass equal to 10 nm). The fluorescence emis- sion was observed through a cutoff filter. Each phase and modulation value represents the aver- age of several measurements. The data were analyzed using the nonlinear least-squares method, and all presented fitting parameters were obtained using frequency-dependent weighting errors [28]. In fluorescence lifetime measurements the enzyme concentration was about 0.5-1.0 m g / m l , the tri- fluoroethanol concentration was equal to 3.4 mM, and the N A D + concentration was 0.8 mM.

Spin-lattice relaxation times for protons in pro- tein solutions were measured at a resonance frequency of 26 MHz, using the spin echo spec-

trometer built in the Institute of Nuclear Physics in Krakbw, Poland. The spin-lattice relaxation times were measured by the Carr-Purcell pulse sequence: 7r-~r/2-Tr, at temperatures varying from about 5 ° C to about 70 ° C. The temperature of the sample was stabilized by a type 650 temperature controller (Unipan, Poland), to an accuracy of + 0.1 ° C. The errors in the T a values were smaller than 5%. In N M R measurements the protein con- centration was 50 m g / m l and the N A D + con- centration was 6.25 raM. In all temperature de- pendent measurements the temperature was raised in steps of a few degrees centrigrade. After the temperature had become constant at a new value, a period of about 10 min was allowed for the sample to reach equilibrium.

Results

The effect of temperature on the quantum ef- ficiency of fluorescence of liver alcohol dehydro- genase alone, as well as of the enzyme's binary complex with N A D +, and its ternary complex with N A D + and the substrate analogue trifluoro- ethanol, was studied in 0.1 M phosphate buffer (pH 7.5).

As described by Kirby and Steiner [15], the temperature dependence of the fluorescence quantum yield, if only one deactivation process is significant, may be expressed as follows:

(1)

where Q is the fluorescence quantum yield at temperature T, k / is radiative rate constant (as- sumed to be independent of temperature, at least in the region between 0 and 70°C), f is a frequency factor of the temperature-dependent deactivation processes, R is the gas constant, E is the activation energy of the deactivation process, and T is absolute temperature. Therefore, a semi- logarithmic plot of ln(Q - 1 - 1) vs. 1/T should give a straight line, whose intercept is In f /k and slope is - E / R . The smooth function of ln(Q a _ 1) vs. 1/T predicted from Eq. 1 reflects the tem- perature dependence of the effective collisions be- tween quenching groups and the fluorophores. For internal residues in globular proteins, the quench-

ing process must involve fluctuations in the pro- tein structure surrounding these fluorophores. Breaks in the plots can be normally expected only when conformational transitions occur and differ- ent deactivation processes take place with signifi- cantly different activation energies.

Plots of ln(Q -1 - 1) against 1/T for each sam- ple of alcohol dehydrogenase in the temperature range of 1 0 - 6 0 ° C are shown in Fig. 1. For the enzyme alone the relationship is linear from 10 to 25 ° C, corresponding to an activation energy of about 1.8 kcal /mol . At about 25°C a thermal transition occurs and a second linear region is observed in the temperature range from 25 to 42 ° C, characterized by an activation energy of about 0.4 kcal /mol . Above 44 ° C, in case of liver alcohol dehydrogenase alone, the plot of ln(Q -1 - 1 ) vs. 1/T departs from linearity. This latter deviation may be reasonably ascribed to the onset of thermally induced denaturation of the protein. The activation energy of this process is approx. 17 kcal /mol . In the presence of ethanol or TFE (the plots are not included in this paper) the enzyme displays both the low (about 25°C) and high (about 44°C) thermal transitions in similar re- gions of temperature as in the case of the dehydro- genase alone.

When N A D + is added to the alcohol dehydro- genase solution, the low-temperature thermal tran- sition is found to be shifted from about 25 ° C, as for the enzyme alone, to 30°C in the binary complex (Fig. 1). For this complex a plot of ln(Q t _ 1) vs. 1 / T is linear from 10 to 30°C, with an activation energy of 1.7 kcal /mol . Above this temperature a new linearity is observed with a very low activation energy, equal to 0.05 kcal /mol . It is known that trifluoroethanol forms a specific alcohol dehydrogenase ternary complex with N A D + [16]. A plot of ln(Q - 1 - 1) vs. 1/T for such a ternary complex is also presented in Fig. 1. In a temperature range from about 10 ° to 60 ° C, a linear relationship is observed, giving an activa- tion energy equal to 0.95 kcal /mol .

The changes in the plot of ln(Q 1 _ 1) vs. 1/T for the enzyme alone, as well as for the enzyme's binary complex, suggest the existence of different temperature-dependent forms of alcohol dehydro- genase. Unfortunately, the steady-state measure- ments presented above cannot give information

213

concerning which of the two tryptophan residues in the enzyme is involved in this transition. To answer this question, we used variable-frequency phase-modulation fluorescence measurements to resolve the intrinsic fluororescence of the enzyme into its two lifetimes. We did this for the enzyme alone as well as its ternary complex. Measure- ments were made as a function of temperature from about 5 to 35 ° C.

Typical frequency-dependent phase and mod- ulation data for the enzyme alone are presented in Fig. 2. At each particular temperature the phase and modulation data were analyzed, using a non- linear least-squares procedure. We analyzed the data in terms of a double exponential with fluores- cence lifetime values, ~'1 and ~-:, as well as their fractional intensities, f l and f2, allowed to run free. At each particular temperature the fits to the experimental data were satisfactory. At 10 ° C, for example, we obtained ~-1 = 6.85 _+ 0.05 ns and ~'2 = 2.95 + 0.06 ns. These values are similar to the values that have been found for liver alcohol dehydrogenase by single photon counting mea- surements [24] and ~'1 and ~'2 can be identified as being the lifetimes of Trp-15 and Trp-314, respec- tively. When the results were plotted in Arrhenius fashion, the plots of log 1/~" i vs. 1/T for the

100

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Fig. 2. Phase and modu la t i on da ta for the in t r ins ic fluores- cence of a lcohol dehydrogenase in 0.05 M phospha te buffer (pH 7.5) at 1 0 ° C . The pro te in was excited at 289 nm, and emiss ion was observed th rough a cutoff filter. The solid l ine

represents a two-componen t fit to the exper imenta l da ta wi th

pa rame te r s ~'1 = 6.85, ~'2 = 2.9, f l = 0.75 and f2 = 0.25.

214

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Fig. 3. (A) Arrhenius plots of l / ? i for Trp-15 (O) and Trp-314 (O) for alcohol dehydrogenase alone. (B) Fraction of fluores- cence intensity for the Trp-314 residue. The analysis was performed in terms of a double exponential with ~1, ~2, f] and f2 allowed to run free.

enzyme alone did not show satisfactory linearity for either Trp-15 or Trp-314 (Fig. 3). This result was not surprising, since it is known that it is very difficult to resolve fluorescence lifetimes values, using either pulsed or harmonic methods, when the ratio of two fluorescence lifetimes is equal to or lower than 2. In Fig. 3 we also show the values of the fraction of the fluorescence intensity due to the Trp-15 residue obtained from these fits. In the temperature range from 5 to 35 ° C, these fraction values are randomly distributed around an aver- age value equal to 0.75 + 0.05. When a nonlinear least-squares analysis was performed, using fixed values of f15 = 0.75 and f314 ~ - 0 . 2 5 , the analysis gave satisfactory fits.

When ~'t5 and "/'314 values are obtained in this manner (i.e., fixing f15) are plotted in Arrhenius fashion, a satisfactory relationship is obtained (Fig. 4). As can be seen in the plot of log 1 / r vs. 1/T for Trp-15, the enzyme alone is linear over the

temperature range from 5 to 35 °C, and is char- acterized by an activation energy equal to 0.82 kcal/mol. The Arrhenius plot for the buried Trp- 314 residue is also linear in the temperature range from about 5 to 25 ° C, with a slope corresponding to an activation energy equal to 1.47 kcal/mol. At 25 °C adeviation from linearity is observed. This result supports the steady-state measurements pre- sented above, and suggests that the thermally in- duced changes observed in the enzyme alone are associated with rearrangement of the protein sub- units. The observed thermally induced transition around Trp-314 is fully reversible. This fact is demonstrated in Fig. 4, which shows that the data obtained after cooling the sample correspond to those obtained by heating the sample.

It is known that the formation of the ternary complex leads to the quenching mainly of buried Trp-314 fluorescence by singlet-singlet energy transfer [23,24]. For comparison, ~'3t4 in the free enzyme and ternary complex is 2.95 and 1.47 ns respectively, at 10°C. ~'15 values in the free en- zyme and ternary complex are 6.85 and 6.60 ns,

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81 - - I I i _ _ 1 3.2 3.3 3,4 3 .5 3.6

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Fig. 4. Arrhenius plots of 1/7" i for Trp-15 ( t , A) and Trp-314 (O, zx) for alcohol dehydrogenase (alone). The triangle repre- sents data obtained by cooling. Nonlinear least-squares analyses were performed with fixed f l = 0.75 and f2 = 0.25 values, and "r], ~'2 were allowed to run free.

respectively, at 10 ° C. The fluorescence lifetime of Trp-314 is much smaller than that of Trp-15 in the ternary complex. We have studied the temperature dependence of '1"314 and %5 in this complex and have analyzed the phase and modulation data, using a nonlinear least-squares procedure, with r~, r 2, and the fractions, f l and f2, allowed to run free. At each particular temperature the fit to the experimental data was satisfactory. The results are plotted in Arrhenius fashion in Fig. 5. In the ternary complex an activation energy of 1.9 kca l /mol and 1.4 kca l /mol are found for Trp-314 and Trp-15, respectively.

The temperature dependence of the intrinsic fluorescence yields presented in Fig. 1 indicates that the thermal transitions in liver alcohol dehy- drogenase depends on its interaction with ligands. To support this suggestion, fluorescence measure- ments were made with the noncovalently bound apolar probe, 2-(p-toluidinyl)napthalene-6-sul- fonic acid. The fluorescence of this probe was measured as a function of temperature, as pre- sented in Fig. 6. TNS is one of a class of com- pounds which are weakly fluorescent in water, but become highly fluorescent upon binding to an apolar site in proteins [17]. When TNS was added

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Fig. 5. Arrhenius plot of l / ' r i for Trp-]5 (e), and Trp-314 (O) for the LADH-NAD+-t r i f luoroe thanol ternary complex.

215

10

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4

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I I 1 I i I 0 10 20 30 40 50 6 0

T e m p e r o t u r e ( ° C )

Fig. 6. Temperature dependence of the fluorescence of TNS bound to alcohol dehydrogenase in 0.05 M phosphate buffer (pH 7.5), when excited at 366 nm. O, alcohol dehydrogenase alone; II, binary complex in the presence of 0.5 mM NAD+; + , ternary complex in the presence of 0.5 m M N A D + and TFE (10 mM). Protein concentration was 0.5 m g / m l and TNS concentration was 63/~M.

to the alcohol dehydrogenase solution, the temper- ature dependence of the former's fluorescence in- tensity revealed thermally induced transitions, oc- curring at about 27°C and 50 °C. The tempera- ture regions of these transitions are in good agree- ment with those which have been found in the above intrinsic f luorescence measurements . Addition of N A D + to the enzyme is in the pres- ence of TNS caused the quenching of TNS fluo- rescence to some degree, and this observation is in agreement with previously published data [18]. The temperature dependences of the fluorescence intensity of TNS in the L A D H - N A D + binary complex, also shown in Fig. 6, exhibits a thermally induced transition at about 35 ° C. This is close to the one found by intrinsic fluorescence measure- ments. In the ternary complex, L A D H - N A D +- TFE, a linear relationship of TNS fluorescence intensity versus temperature is observed over the range of 5-60 ° C.

The temperature dependence of the proton spin-lattice relaxation times, T1, of fiver alcohol dehydrogenase and its binary complex with N A D + are presented in Fig. 7. For the enzyme alone, In T 1 is a linear function of T-1 in the temperature range from about 4 ° C up to about 52°C. The activation energy calculated for this temperature-

216

60 40 20 T ( °C) I I I i ~ t

8.5

80 ~

7 5 -

3.0 3.2 3.4 3 6

I / T x 103 (K -1)

Fig. 7. Temperature dependence of the proton relaxation time, T 1, of alcohol dehydrogenase in 0.05 M phosphate buffer (pH 7.5). O, alcohol dehydrogenase alone; o, alcohol dehydro- genase binary complex in the presence of 6.25 mM NAD+: A, relaxation time T l, for 0.05 M phosphate buffer (pH 7.5). Protein concentration was 50 mg/ml.

dependent process is equal to 4.35 kcal/mol. The observed departure from the linearity above 52 °C probably indicates the protein denaturation processes.

As can be seen in Fig. 7, the binding of NAD + to alcohol dehydrogenase causes a decrease in the spin-lattice relaxation times T 1 in the nonde- naturating temperature range. This observation supports the idea that in a solution the enzyme undergoes conformational changes upon binding NAD +.

For the binary complex, L A D H - N A D +, In T 1 is a linear function of 1/T up to about 62 ° C (Fig. 7). However, the slope of the curve is significantly different from that obtained for alcohol dehydro- genase alone. The activation energy calculated for the binary complex is equal to 3.8 kcal/mol. It should be noticed that in the binary complex the departure from linearity of the plot occurs at a much higher temperature than in the enzyme alone (Fig. 7). This observation remains in agreement with an earlier report [19] that the binding of NAD + protects alcohol dehydrogenase against thermal inactivation.

Discussion

It is now well established that changes in the quantum yield, lifetime and other characteristics of the intrinsic fluorescence of proteins can be used to monitor their conformational changes and their interaction with ligands [20]. The results of fluorescence studies presented in this paper pro- vide further details on the functional properties of liver alcohol dehydrogenase, which may reflect the dynamic behavior of the enzyme under different conditions, such as the presence and absence of ligands, and variation of temperature.

The intrinsic fluorescence of the enzyme, when excited at 290-295 nm, arises almost entirely from the two types of tryptophan, Trp-15 and Trp-314, in this protein. The fluorescence of the dehydro- genase provides a very sensitive means for study- ing the protein's structure and its changes. The temperature dependence of the tryptophanyl fluo- rescence properties can reflect the dynamics (nanosecond time scale) of the protein structure surrounding the tryptophan residues. It can be expected that plots of ln(Q 1_ 1) or log(1/ri) versus 1/T should be a linear function with no breaks [15,7]. The nonlinear plot of ln(Q l _ 1) vs. 1/T for alcohol dehydrogenase suggests that at about 25°C a thermal transition occurs between two different temperature-dependent forms of the enzyme. The fluorescence lifetime data in Fig. 4 further suggest that this thermal transition prim- arily affects Trp-314, the internal tryptophan re- sidue. The thermal transition is shifted to higher temperature by the binding of NAD +. Upon for- ming the NAD+-trifluoroethanol ternary complex, the thermal transition is apparently eliminated.

The values of the activation energy calculated from the data presented in Figs. 1, 5 and 6 are much lower than the value of 6-8 kcal/mol found for tryptophan in water [15]. They are also lower than the values of 2.5 and 4 kcal /mol which have been found for a few proteins studied so far [7].

When the temperature dependence of the tryp- tophan fluorescence of single tryptophan contain- ing proteins, such as azurin [21] and parvalbumin [29], were studied, an activation energy of 0.8 to 1.5 kcal /mol was found. In these proteins a single tryptophan residue, like the Trp-314 in liver al- cohol dehydrogenase, is located in a very hydro-

phobic, nonpolar environment and is inaccessible to aqueous solvent [21,29]. These observations may lead to the conclusion that the activation energy for the temperature-dependent deactivation pro- cess in proteins can depend on the environment of the tryptophan residues. The fluorescence lifetime measurements performed at various temperatures in this paper indicate, however, that both Trp-314 and Trp-15 in alcohol dehydrogenase have rela- tively small activation energies for the tempera- ture-dependent deactivation process. In fact a slightly higher activation energy (between 5 and 25 °C) is found for the buried Trp-314, in com- parison to exposed Trp-15.

The thermal behavior of alcohol dehydrogenase was also monitored independently by measuring the fluorescence intensity of the apolar probe, TNS. The results presented in Fig. 6 show that in the enzyme alone, as well as in the binary complex with NAD +, the thermally induced conforma- tional changes, observed in the nondenaturing range of temperature, are in good agreement with those found when observing the intrinsic fluores- cence quantum yields (Figs. 1 and 4). It is known that TNS, which has a negative charge attached to the ring system, binds at the adenosine site of the dehydrogenase molecule [1]. The thermally in- duced changes observed here, both in the enzyme alone and in its binary complex, may involve not only the environment of Trp-314, but also prob- ably a larger part of the nucleotide-binding do- main of alcohol dehydropenase. The binding of NAD + and trifluoroethanol to the enzyme-TNS complex apparently eliminates the transition; this observation is in agreement with results presented in Fig. 1.

The temperature dependence of the water spin- lattice relaxation time of the enzyme alone, as well as of its binary complex with NAD + (presented in Fig. 7), shows a linear relationship up to the temperature at which denaturation probably oc- curs. The binding of NAD + to the enzyme causes a decrease in the spin-lattice relaxation time, T a. The decrease in T, after the binding of NAD + to the enzyme can result from a change in the num- ber of bound water molecules as well as from changes in the environment of bound water mole- cules resulting from a conformational changes in the protein. This suggestion is supported by X-ray

217

diffraction studies which indicate that in the pres- ence of coenzyme the enzyme molecule undergoes a large conformational change from an open to a closed form. This conformational change is accompanied by the displacement of some water molecules from the coenzyme binding site [5]. It has been suggested that liver alcohol dehydro- genase exists as an equilibrium mixture of both conformations in the absence of coenzyme with the equilibrium favoring the open conformation. However, in ternary complexes the equilibrium is shifted towards the closed conformation.

Further, it is tempting to suggest that in solu- tion the thermal transitions observed in alcohol alone, as well as in its binary complex with NAD +, may results from a small reorientation of the enzymes subunits and that such a reorientation may be characteristic to the enzyme's action. In fact, the thermal transitions in alcohol hydro- genase are very similar to those found in other proteins such as thiosulfate sulfurtransferase [8,27] and yeast hexokinase [9,27]. These enzymes also consist of two lobes separated by a deep cleft in which substrates are bound.

Acknowledgements

This research was partially supported by grant R. III. 13 from the Polish Ministry of Science, Higher Education and Technology to Z.W., and by National Science Foundation Grant DMB- 8511569 to M.R.E.

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