Eur. J. Biochem. 248, 80-85 (1997) 0 FEBS 1997

Dynamic structure/function relationships in the a-chymotrypsin deactivation process by heat and pH Pedro LOZANO, Teresa DE DIEGO and Jost Luis IBORRA

Departamento de Bioquimica y Biologia Molecular B e Immunologia, Facultad de Quimica, Universidad de Murcia, Spain

(Received 2 May/9 June 1997) - EJB 97 063914

The activity decay of a-chymotrypsin due to temperature and pH has been related to the associated structural changes of the protein that could be studied with the help of melting temperature which was determined by using ultraviolet absorption spectroscopy, and fluorescence spectra measurements. The kinetic behaviour in activity loss of a-chymotrypsin followed a two-step deactivation model, involving an intermediate state. The increase in temperature and pH showed a clear deactivation effect, reducing exponentially the half-life of the enzyme. At pH 7.0, this two-step deactivation process was also observed with both the maximum fluorescence intensity (Imax) and emission wavelength (Amdx). The series-type kinetic model allowed to establish a clear correlation between the activity and the fluorescence spectral normalized data: the intermediate state of the enzyme occurred at an identical deactivation denaturation level (a,), and a proportionality between the decay rate constants was observed. As a function of the incubation temperature, another correlation was observed between the a , profile, initial A,,,,, and thermal unfolding transition, allowing to identify the intermediate state of the kinetic model as that obtained at the melting temperature (43.9"C).

Keywords: kinetic analysis ; denaturation; fluorescence properties ; protein unfolding ; serine protease.

Understanding the molecular mechanism of the deactivation of enzymes is still an unresolved problem of applied biocata- lysis. The analysis of the enzyme deactivation process and the possibility of enhancing thermal stability is an important con- sideration in designing continuous systems for the development of biotechnological process to an industrial scale [l-31.

The serine-protease a-chymotrypsin is one of the most ana- lyzed enzymes in the studies of the stabilization mechanism due to the large number of potential applications for peptide synthe- sis in unconventional media [4-61. In this way, concepts that have proved to be useful in protein thermostability may be ap- plied to achieve the goal of rational enzyme stabilization in nonaqueous solvents [3].

Different strategies to increase the a-chymotrypsin stability have been used and described in the literature: addition of low- molecular-mass compounds (i.e. polyols [7] and salts [S]), hy- drophilization of the external surface by chemical modification [9], enzyme immobilization onto insoluble supports [6, 101 and entrapment into reverse micelles [ll]. In many cases, such ap- proaches to enzyme stabilization have been empirical [12], while in other different explanations have been offered about the modi- fication in enzyme stability, such as changes in the structure of water [7, 131, elimination of incorrect protein refolding by the increase i n hydrophilicity [4, 8, 91, or the maintenance of the water shell around the protein molecule [4, 61. For some of these 16-9, 141 the stability of native or modified a-chymotrypsin

Correspondence to J. L. Iborra, Departamento de Bioquimica y Bio- logia Molecular B e Immunologia, Facultad de Quimica, Universidad de Murcia, P.O. Box 4021, E-30001 Murcia, Spain

Fax: +34 68 36 41 48. E-mail: jliborra@fcu.um.es URL: http ://www.um.es Abbreviation. AcTyrOEt, N-acetyl-L-tyrosine ethyl ester. Enzyme. a-Chymotrypsin (EC 3.4.21.1).

have been analyzed from a mechanistic point of view, proposing a two-step deactivation model (E,-+E,-+E,) to explain kinetic and thermodynamic results. According to this model, the native state (EN) unfolds to the intermediate state (E,), and is followed by inactivation to give the denaturated state (ED.) The multipha- sic nature of the enzyme molecule during a deactivation process cuggests that different internal events take place in its conforma- tional transitions [ l]. In this order, some complementary experi- ments related to structural modification of the enzyme unfolding during the deactivation process have been performed to confirm this hypothesis. Thus, Owusu and Berthalon [I41 observed a break in the enzyme melting or unfolding temperature in the Arrhenius plot related to the intermediate state. In the same way, Owuzu and Berthalon [15], and Levitsky et al. [I61 have shown a zig-zag behaviour in the temperature-dependent rate constant. Mozhaev et al. [9] have correlated empirically the kinetics of enzyme thermal deactivation with some changes in the fluores- cence spectral properties of the enzyme, such as the increase in the maximum wavelength and the decrease in the maximum intensity in aqueous media.

It can be assumed that a decay in enzyme activity during a deactivation process should be in direct correlation with simulta- neous conformational changes of the protein. However, in many cases, systematic and quantitative analysis of enzyme deactiva- tion and protein unfolding relationships in a-chymotrypsin have been made. The aim of this work was to study the a-chymotryp- sin stability in aqueous media as a function from both kinetic and conformational points of view. Simultaneous intrinsic fluo- rescence and thermal unfolding transition studies have been made during the protein unfolding process, and quantitatively correlated with the enzyme activity loss. An overall two-step kinetic model of enzyme deactivation/unfolding was used to in- tegrate all results and to emphasize the dynamic structure/func- tion relationships of a-chymotrypsin.

Lozano et al. (EUK J. Biochem. 248) 81

MATERIALS AND METHODS

Materials. a-Chymotrypsin type I1 from bovine pancreas was purchased from Sigma Chem. Co. N-Acetyl-L-tyrosine ethyl ester (AcTyrOEt, Sigma Chem. Co.) was used as standard sub- strate. All remaining reagents were analytical grade and used without additional purification.

Assay of enzyme activity. The esterase activity of a-chymo- trypsin was determined by the pH-stat method described in de- tail by Wilcox [17]. A video-tritrator VIT-90 equipped with an autoburette (ABU 91) and a sample station SAM YO (Radiome- ter) was used. The protocol was as follows: a 3-ml sample of 50 mM AcTyrOEt in 30% (madvol.) aqueous ethanol solution, containing 20 mM CaC1, were placed into a reaction vessel ther- mostated at 40°C. The reaction was started by addition of 50 pl 0.1 % (masdvol.) a-chymotrypsin aqueous solution and the pH was maintained constant at 7.0 by continuous addition of 50 mM NaOH, as titrant. One unit (U) of activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 pmol AcTyrOEVmin under standard conditions of assay (pH 7.0, 40°C).

Study of enzyme stability. a-Chymotrypsin solutions (0.4 mg/ml) in 10 mM sodium phosphate pH 6.0, 6.5, 7.0, 7.5 or 8.0, were incubated at 25, 30, 35, 40, 43,45, 50 or 60°C. At regular intervals of time, 5O-pl aliquots were extracted from the incubation mixture and the residual activity was determined im- mediately as described above. The results obtained were fitted to model theoretical curves using a non-linear regression pro- gram of iterative convergence with the Marquardt-Levenberg al- gorithm method included into the Sigmaplot. Scientific Graphic System version 5.01 (Jandel Scientific Co.)

Fluorescence studies. Fluorescence spectra of a-chymotryp- sin were monitored on a Hitachi spectrofluorometer (model F- 4500) in the temperature range 30-60 "C, using a thermostated cell (+OS"C). 10 mM sodium phosphate pH 7 (2.5 ml), was previously incubated in the cell for 15 min and then 0.5 ml a- chymotrypsin (0.06 mg/ml) was added, starting the fluorescence spectrum record (excitation wavelength, 295 nm ; scanning rate, 1200 n d m i n ; bandwidth, 5 nm). The fluorimeter automatically supplied corrected spectra, by comparison with a 1 nM standard solution of rhodamine B in glycerol, to avoid the changes in lamp output and the instrument geometry [18]. The maximum emission wavelength of the sample was determined as the wave- length for which dl/d;l = 0.

Thermal unfolding transition. The thermal denaturation of a-chymotrypsin was measured in 10 mM sodium-phosphate at different pH (6.0, 6.5, 7.0, 7.5 and 8.0), using a GBC-918 visi- ble/ultraviolet spectrophotometer equipped with a Pletier-effect temperature-controlled base. The thermal transitions were fol- lowed by the decrease in absorbance at 281 nm (5-nm band- width), with a resolution of 7-9 data points/"C. The sample (a 0.3-mg/ml chymotrypsin solution) was previously incubated at 30°C for 10 min and then, heated by the Peltier-effect system up to 75"C, at a rate of 0.3"C/min, which was measured by temperature probes. The temperature at the midpoint of thermal denaturation or melting temperature (t,,,) was calculated at the minimum of the first derivative curve of thermal denaturation obtained by the software application of the system.

RESULTS AND DISCUSSION

Deactivation kinetic model. According to the classification of enzyme deactivation mechanisms proposed by Wada et al. [IY], complex enzyme deactivations can be subdivided in two cate- gories : (a) deactivations involving one stable intermediate (three-state mechanism) and (b) deactivations involving more

400 - 300 5

1.0

0.8 F

200 g 0.2 100 8

v

E '5 0.6

8 0.4 .- U

.- U

n n 0 - 0 20 40 60 20 30 40 50 60

Time ( min. ) Temperature ( 'C )

Fig. 1. Influence of temperature on activity and stability of a-chymo- trypsin. (A) Thermal deactivation of a-chymotrypsin in 10 mM sodium phosphate pH 7.0 at 30°C (O), 40°C (A), 43°C (O), 45°C (N), 50°C (0) and 60°C (A). Residual activity of a-chymotrypsin was measured at 40°C and pH 7.0, using 50 mM AcTyrOEt as substrate. (B) Effect of temperature on the esterase activity of a-chymotrypsin at pH 7.0, mea- sured at the temperatures plotted on abscissa.

than one stable intermediate (four- or higher-state mechanisms). Therefore, it is reasonable to expect that more than one type of bond must be broken before the enzyme deactivation occurs, and should be followed by different conformational changes of the enzyme. However, the predominance of a second stage of transi- tion has never been considered because of the lack of sensitivity of the experimental measurements (i.e. calorimetry), as well as the lack of any adequate response of the three-state mechanism to explain the kinetic behaviour of enzyme deactivation [20].

A series-type kinetic model of protein unfolding has been considered to analyze the a-chymotrypsin deactivation [20]. This model involves two first-order steps, with one active pre- cursor and a final enzyme state with possible non-zero activity, as follows.

N- CI-U (1)

where N, CI and U are the native, intermediate and unfolded homogeneous enzyme states, respectively, which exhibit dif- ferent specific activities; a1 and a, are the ratio of specific activi- ties CI/N and U/N, respectively, and k , and k, are first-order deactivation rate constants (min-I). Considering as initial condi- tions that, N, = = No, and el,=, = U,=, = 0, the specific activ- ity of each enzyme form can be determined by the integration of the deactivation rate equation of each step (-diV/dt = k , N ; - dCI/dt = k,CI - k,N).

The fractional activity remaining, a, is given by a weighted- average function of specific activities of the different enzyme state yields, as follows:

kl k2

0, I

N + a , . CI + a,. U

NO a =

= a, + [1 + a,k , / (k , - k , ) - a,k,/(k, - k , ) ]

. lexp ( - k l . tll - (a , - a,)[k,/(k, - kl)l

. [exp (- k2 . t)] .

( 2 )

The experimental enzyme deactivation data were modeled by Eqn (2) using a non-linear regression procedure based on the Marquardt-Levenberg method of iterative convergence to calcu- late all deactivation parameters. The values of k , , k2, a, and a, were constrained to be non-negative, and the theoretical deacti- vation curves were obtained from Eqn (2) by substitution of the calculated convergence values. A good agreement between the experimental and theoretical data was considered when the cor-

82 Lozano et al. ( E m J. Biochem. 248)

Table 1. Deactivation kinetic constants (a,, a,, k , and k,) and half- life (tm) of a-chymotrypsin in 10 mM sodium phosphate pH 7.0, as a function of the incubation temperature.

Temperature a, a2 k , lO-'x k, t,,,

"C min-' min

30 0.52 0 0.015 0.4 235.1 40 0.48 0 0.08 6.0 26.5 43 0.30 0 0.20 25.0 5.8 45 0.05 0 0.24 30.0 3.1 50 0.01 0 0.50 80.0 1.4 60 0.001 0 1.50 100.0 0.4

relation coefficient was higher than 0.98 for each deactivation experiments.

Kinetic analysis of the a-chymotrypsin thermal deactivation. The influence of temperature on a-chymotrypsin stability has been studied between 30-60"C, using 10mM sodium phos- phate pH 7.0 as media. Fig. 1 A shows the deactivation profiles, depicted by the experimental points, of a-chymotrypsin at each assayed temperature, while Fig. 1 B shows the influence of tem- perature on the esterase activity of the enzyme. In all cases, the depicted curves of Fig. 1A are theoretical, and were obtained from the experimental data by using the series-type deactivation model (see Eqn 2). The calculated values of the deactivation parameters (a,, a,, k , , and k,) are included in Table 1. As can be seen, even if the maximum activity of a-chymotrypsin was ob- tained at 40"C, the increase in the incubation temperature from 30°C yielded a continuous decrease in the enzyme stability, showing the high sensitivity of the enzyme towards thermal con- ditions. Moreover, it is remarkable that the greatest loss of en- zyme activity occurred between 4O-5O0C, which is precisely the range where an enhancement of the deactivation process could be observed. These phenomena could be quantified by analysis of the deactivation kinetic constants (see Table 1). In all cases, the a, parameter was lower than 1 , showing the loss of enzyme activity during the transition to the intermediate state CI. This parameter remained practically unchanged between 30-40°C, and then continuously decreased to a value very close to zero. The most important changes occurred between 43- 45°C. Regarding the a, parameter, it remained 0 in all cases indicating that the final state of a-chymotrypsin (U) is a fully deactivated state, as has been experimentally proved at long de- activation times. In addition, the deactivation rate constants of both steps ( k , and k,) increased exponentially with the incuba- tion temperature, k , being always 10-fold higher than k,. This biphasic nature of the deactivation process is clearly observed in the convexity of the curves towards the origin. As a matter of fact, the increase in the incubation temperature produced an enhancement of the convexity of the curves, as well as an expo- nential decrease in the half-life of the enzyme.

As it was previously reported [16], bimolecular processes (aggregation or autolysis) were avoided under all conditions studied, because the thermodeactivation kinetics for a-chymo- trypsin did not depend on the assayed enzyme concentration.

Owusu and Berthalon [14, 1.51 have analyzed the stability of a-chymotrypsin from both the kinetic and thermodynamic points of view, during a short interval of time (less than 10 min) at acidic pH (5 -6). Under these conditions, the kinetic analysis of experimental data should be adequately represented by only one deactivation rate constant, obtaining a single slope in the Arrhenius plot (In k versus l/n. However, these authors have

0.56

0.55

0.54

0.53

0.52

0.51

0.50

0.49 -5 30 35 40 45 50 55 60 65

Temperature (OC )

Fig. 2. Thermal unfolding curve of a-chymotrypsin (0.3 mg/ml) in 10 mM sodium phosphate pH 7.0. Initial temperature, 30°C; final tem- perature, 70°C; heat rate, 0.3 "C/min.

- 1 .o

0.8 7 400 ~

a 3 a '5 0.6 Y

J

2 0.4 200 :z CI

0 2 Y 0.0 0

0 20 40 60 4 6 8 10

Time ( min. ) PH

Fig. 3. Effect of pH on the a-chymotrypsin at 40°C. (A) Deactivation profiles at pH 6.0 (O) , 6.5 (O), 7.0 (A), 7.5 (0) and 8.0 (W). (B) Ester- ase activity pH profile of a-chymotrypsin, measured at the pH plotted on the abscissa. Activity of a-chymotrypsin was measured at 40°C, using 50 mM AcTyrOEt as substrate.

observed biphasic behaviour in this Arrhenius plot when the de- activation conditions were enhanced (i.e. by the presence of 1 M guanidinium chloride), which suggests a two-step deactivation mechanism. Additionally, this break in the Arrhenius plot was related to the unfolding or melting temperature (tm) of a-chymo- trypsin, a parameter that measures indirectly the conformational stability of a protein.

Fig. 2 depicts the heat unfolding of a-chymotrypsin at the maximum wavelength (281 nm) in 10 mM sodium phosphate pH 7.0. The ultraviolet spectral changes have previously been related to the exposure of buried tryptophan and tyrosine resi- dues from the non-polar interior of the protein to the polar envi- ronment of the bulk solvent [21]. As can be seen in Fig. 2, the enzyme was unfolded continuously with heat, to an absorbance value similar to that observed in denaturing conditions (8 M urea). From this curve, a t,,, value of 43.9"C was determined as the minimum of the function from the first derivative curve, a value of the same order as that obtained by other authors [14, 151. These results could be related to both the enhancement of the thermal deactivation process from 40°C to 50"C, as men- tioned before in Fig. 1, and the decrease in the a, parameter obtained between 43-45°C (see Table 1). The protein unfolding transition from the native state (N) to the fully unfolded state (U) should occur via a stable conformation that is neither fully folded nor fully unfolded, so-called by Creighton [22] the com- pact intermediate (CI) of the molten-globule-state.

Lozano et al. (Eur: J. Biochem. 248) 83

Table 2. Deactivation kinetic constants (a,, a,, k , and k,) and half- life (&) at 40°C of temperature, and melting temperature (tm) of a-chymotrypsin in 10mM sodium phosphate as a function of the incubation pH.

PH a, a2 k, 10-3xk2 t,,* t,

min-' min "C

6.0 0.80 0 0.04 3.0 182.7 47.1 6.5 0.77 0 0.05 4.0 128.9 46.2 7.0 0.48 0 0.08 6.0 26.5 44.0 7.5 0.45 0 0.13 30.0 11.8 43.6 8.0 0.40 0 0.30 50.0 4.8 42.0

300 350 400

h emission ( nm )

342

340 - 338 E

334 r̂

- 336 2

4

- 332

330 0 20 40 60

Time ( min )

- Fig. 4. Fluorescence characteristic of a-chymotrypsin. (A) Fluores- cence spectrum in 10 mM sodium phosphate pH 7.0 at 40°C. (B) Evolu- tion of the maximal fluorescence emission intensity (0) and maximal emission wavelength (A) of a-chymotrypsin with time, in 10mM sodium phosphate pH 7.0 at 4OoC, using an excitation wavelength of 295 nm. For other experimental conditions, see Materials and Methods.

Effect of pH on the a-chymotrypsin stability. The influence of pH on the a-chymotrypsin stability has been studied by using 10 mM sodium phosphate pH 6.0-8.0 as incubation media at 40°C. Fig. 3 A shows the deactivation profiles of a-chymotryp- sin at the assayed pH (data points) and the theoretical curves obtained by using the series-type kinetic model. In this case, it is also remarkable how the increase in pH from 6.0 to 8.0 (a value where the maximal enzyme activity was obtained, Fig. 3 B) produced an enhancement of the deactivation process, as can be seen by the increase in the convexity of curves in Fig. 3A. Moreover, the deactivation kinetic parameters (see Ta- ble 2) showed an evolution that was according to the enhance- ment of the deactivation process with pH alkalinization: a decrease in the a, parameter and, an increase in both k , and k,, rate constants. Thus, increasing the alkalinity of the media re- sulted as well in lowering the enzyme stability, which was indi- cated by a continuous reduction of both the half-life and the t , values. Obviously, the increase in pH from 6.0 to 8.0 (to a value near to the isolectric point, 8.5 [23]) produced the deprotonation of strong basic groups (amino and guanidinium) of lateral chains of lysine and arginine residues, reducing the overall positive charge of the protein. These facts should be related to changes in electrostatic interaction that are involved in the maintenance of the native conformation of the enzyme, by reducing the over- all unfolding energy [22 ] . Nevertheless, the alkaline deactivation of the enzyme is lower than the thermal deactivation: the loss

1. a e

0.2 - 0.0

0 20 40 60

Time ( min )

Fig. 5. Deactivation and unfolding profiles of a-chymotrypsin, mea- sured as normalized activity (a, M), normalized maximum fluores- cence intensity (1, 0) and normalized maximum emission wavelength ( I , A) parameters, in 10 mM sodium phosphate pH 7.0, with time.

in stability produced by the increase in 10°C is much higher than that observed with increase in pH of 1.

Fluorescence analysis of the enzyme deactivation. It is well known [4, 8 - 111 that protein denaturation causes a great change in maximal intensity of fluorescence (Imax) and a red-shift of maximal emission wavelength (Amax) both resulting the increase in polarity of the microenvironment of the tryptophan residues of the protein globule. Fig. 4A shows the spectra of native a- chymotrypsin (A,,, = 330.8 mn), while Fig. 4B depicts the evo- lution of both the I,,, and the /1,,,,, parameters of a-chymotrypsin with time at pH 7.0 and 40°C. As can be seen, I,,,,, was continu- ously reduced with time, showing a kinetic profile similar to that observed in Figs 1A and 3A under the same environmental conditions, while A,,,,, shows a hyperbolic increase. Thus, under denaturing conditions such as 8 M urea, the absolute value of I,,,, was reduced threefold while /I,,,,, increased from 330.8 nm to 347 nm. A kinetic analysis of these experimental data has been carried out using the proposed series-type kinetic deactiva- tion model to try and correlate them to the deactivation process of the enzyme.

The fluorescence parameters (I,,,,,, A,,,,) could be considered as good conformational parameters for the protein: they gave a measurement of the dynamic shift of the conformation from the native to the unfolded state occurring during the deactivation1 denaturation process of a-chymotrypsin. In the same way as that used to describe the activity (Eqn l), kinetic equations could be proposed :

and

(3)

(4)

It was assumed that I , , Ic,, Zu are the maximum fluorescence intensity of the native, intermediate and unfolded homogeneous enzyme states, respectively, while AN, Ac,, A, are the maximum emission wavelength of these states, where each state exhibits different specific fluorescence parameters. Thus, a,, and a2 were defined as the ratio of, Ic,/IN, IulIN; and AcJA,, AulAN, respective- ly; while k, and k2 are first-order deactivation rate constants (min-l), respectively for each equation. However, to use the pro- posed series-type kinetic model, normalized and dimensionless

84 Lozano et al. ( E m J. Biochern. 248)

Table 3. Kinetic deactivation constants of dimensionless structure/ function parameters (a, activity; I , fluorescence intensity ; and I , emission wavelength) of a-chymotrypsin in 10 mM sodium phos- phate pH 7.0 at 40°C.

Parameter a, a2 k , X k,

min '

U 0.48 0 0.08 6.0 1 0.48 0 0.16 12.0 1 0.48 0 0.04 3.0

0.5

0.4 d

0.3 - 0 -

0.2

0.1

0.0

334

333 - E

332

330 30 35 40 45 50 55 60 65

Temperature ("C )

Fig. 6. Relationships between the thermal unfolding (-), the a, parameter of the activity decay (0) and the initial maximum emis- sion wavelength (A) of a-chymotrypsin with temperature in 10 mM sodium phosphate pH 7.0.

fl uorescence parameters (intensity, I , and emission wavelength, 1) should be used and can be defined as follows,

and

where lo and & are the initial absolute values of the fluorescence parameters. I, and i, are the absolute values at time t and I, and 2 , are the absolute values at the unfolded state. Then, a weighted-average function, similar to the one used for the spe- cific activities of the different enzyme state yields (see Eqn 2), can be applied to model the normalized and dimensionless fluo- rescence parameters.

Fig. 5 shows the experimental points for all normalized pa- rameters, i.e. activity, fluorescence intensity and emission wave- length. The theoretical deactivatiodunfolding curves were ob- tained by Eqn (2), using the, same method of iterative con- vergence as that used previously for deactivation data [7, 201. The suitability of the model was indicated by the good agreement between the experimental and theoretical data, where a correlation coefficient higher than 0.97 was obtained for all data. The calculated values of deactivation constants for all the assayed activity and fluorescence parameters are included in Ta- ble 3.

The a, constant remained unchanged for all the assayed pa- rameters, showing clearly how the conformational transition of the native state of the enzyme (N) to the intermediate one (CI) occurs simultaneously to the activity changes. Additionally, in all cases a2 was equal to 0, indicating that U is a fully deacti-

vated and unfolded state. However, even if the deactivatiodun- folding k , and k, parameters were not identical, they showed proportionality relationships for both steps : the intensity fluores- cence decay was double the deactivation rate and four times the emission wavelength decay.

These results contribute to confirming a direct and quantita- tive relationship between the structural and functional modifica- tions happening during the deactivation/unfolding process in a- chymotrypsin ; the conformational transition of the enzyme from the native to its unfolded state could be directly related to the deactivation process.

A comparative analysis of data has been performed in order to integrate the influence of the temperature on the conforma- tional/activity transition of a-chymotrypsin. Fig. 6 shows simul- taneous evolution with temperature, at pH 7.0, of various charac- teristic parameters: the a, activity constant (Tables 1 and 2), the initial fluorescence emission wavelength and the native-enzyme fraction, determined from the thermal unfolding transition ex- periment (Fig. 2). All parameters showed exponential profiles with similar behaviour, having a cross-point at 43.9 "C, which was in accordance with the t , value.

The remarkable suitability of the model obtained for three different parameters to describe the dynamic structure/function relationships of a-chymotrypsin should be pointed out. From a kinetic point of view, the unfolding transition (native fraction curve of Fig. 6) involves the evolution of the enzyme conforma- tion from the native state to the fully unfolded state. At this point, the enzyme deactivation process can be considered as a continuous conformational transition which can occur because of the opening of the protein structure. This phenomenon can be simultaneously observed by a red-shift of the emission wave- length due to an increased exposure of tryptophan residues to a more polar environment; it happens simultaneously with a decrease in the specific activity level of the intermediate state (CI). These results clearly show how the numerous and complex events that can take place during an enzyme thermodeactivation process can be observed with the help of parameters describing some structure/function relationships. Such an approach to the phenomenon leads one to conclude that many different compact intermediates constitute a family of states in rapid equilibrium with each other [20]. So, the a, profile should be seen as the evolution of the specific activity of these compact intermediates during a continuous thermal unfolding process with increase in temperature.

However, from a practical point of view, a kinetic model with many different intermediates to explain enzyme deactiva- tion with time at constant temperature, would involve more pa- rameters, making their estimation more difficult but also more reliable. In that sense, the adequate suitability of the proposed two-step kinetic model has been clearly demonstrated. Accord- ing to this model, the presence of native (EN), intermediate (CI) and unfolded (E,) enzyme states appearing during the deactiva- tion process with time at constant temperature (Fig. IA), could be attributed to three different classes of temperature for the thermal unfolding process by continuous increase in temperature (Fig. 6), as respectively less than 30°C, t,, and more than 65°C.

CONCLUSIONS

The study of a-chymotrypsin deactivation by heat and pH followed a two-step kinetic model, where the deactivation pa- rameters were highly dependent on the environmental condi- tions. The enzyme deactivation process was accompanied by substantial unfolding of the protein molecule. This was demon- strated by using fluorescence spectroscopy and thermal unfold-

Lozano et al. (Eul: J . Biochem. 248) 85

ing studies. The simultaneous kinetic analysis of both enzyme deactivation and protein denaturation processes indicated signif- icant correlation between all deactivation parameters : (a) iden- tical a, and proportional rate constant decay (Table 3); (b) clear temperature dependence of the activity decay, measured by a, (Table l), and (c) changes in protein conformation, measured by the unfolding thermal analysis and the red-shift of the emission wavelength.

All the results clearly showed how the a-chymotrypsin activ- ity could be related to the conformation of the protein, empha- sizing the simultaneous deactivation and unfolding processes from the native/active state. In this way, all enzyme stabilization approaches, to be used for enzymatic processes in non-conven- tional media, should be based on the maintenance of the native conformation of the native state of the protein.

This work was partially supported by the Comisidn Interministerial de Ciencia y Tecnologia (grant BI096-1016-C02-01),

REFERENCES 1. Mozhaev, V. V. (1993) Mechanism-based strategies for protein ther-

mostabilization TIBTECH, Trends Biotecknol. 11, 88-95. 2. Gupta, M. N. (1992) Enzyme function in organic solvents, Eur: J.

Biochem. 203, 25-32. 3. Amold, F. H. (1990) Engineering enzymes for non-aqueous

solvents, Trends Biotechnol. 8, 244-249. 4. Mozhaev, V. V., Khmelnitsky, Y. L., Sergeeva, M. V., Belova, A. B.,

Klyachko, N. L., Levashov, A. V. & Martinek, K. (1989) Catalytic activity and denaturation of enzymes in watedorganic cosolvent mixtures: a-chymotrypsin and laccase in mixed watedalcohol, water/glycol and watedformamide solvents, Eur: J. Biockem. 184,

5. Lozano, P., Diego, T. & Iborra, J. L. (1995) Effect of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by immobi- lized a-chymotrypsin, Biotechnol. Lett. 15, 603 -608.

6. Lozano, P., Diego, T. & Iborra, J. L. (1996) Influence of water- miscible aprotic solvents on a-chymotrypsin stability, Biotechnol.

7. Lozano, P., Combes, D. & Iborra, J. L. (1994) Effects of polyols on alpha-chymotrypsin thermostability : A mechanistic analysis of the enzyme stabilization, J. Biotecknol. 35, 9- 18.

8. Levitsky, V. Y., Panova, A. A. & Mozhaev, V. V. (1994) Correlation of high-temperature stability of a-chymotrypsin with ‘salting-in’ properties of solution, Eul: J. Biochem. 219, 231-236.

597 - 602.

Prog. 12, 488-493.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22. 23.

Mozhaev, V. V., Melik-Nubarov, S., Levitsky, V. Y., Siksnis, V. A. & Martinek, K. (1992) High stability to irreversible inactivation at elevated temperatures of enzyme covalently modified by hydro- phillic reagents : a-chymotrypsin, Biotechnol. Bioeng. 40, 650- 662.

Mozhaev, V. V., Sergeeva, M. V., Belova, A. B. & Khmelnitsky, Y. L. (1990) Multipoint attachment to a support protects enzyme from inactivation by organic solvents : a-chymotrypsin in aqueous solutions of alcohols and diols, Biotechnol. Bioeng. 35, 653-659.

Dorovska-Taran, V. N., Veerger, C. & Visser, A. J. W. (1993) Com- parison of the dynamic structure of a-chymotrypsin in aqueous solution and in reversed micelles by fluorescent active-site prob- ing, Eur: J. Biochem. 211, 47-55.

Schmid, R. D. (1979) Stabilized soluble enzymes, A ~ L : Biochem.

Back, J. B., Oakenfull, D. & Smith, M. B. (1979) Increased thermal stability of proteins in the presence of sugars and polyols, Bio- chemistry 18, 5191 -5196.

Owusu, R. K. & Berthalon, N. (1993) A test for the two-stage ther- modeactivation model for chymotrypsin, Food Chem. 48, 231 - 235.

Owusu, R. K. & Berthalon, N. (1994) Determination of enzyme global thermostability from equilibrium and kinetic analysis of heat inactivation, Food Chem. 51, 15-20.

Levitsky, V. Y., Melik-Nubarov, S., Siksnis, V. A., Grinberg, V. Y., Burova, T. V., Levashov, A. V. & Mozhaev, V. V. (1994) Revers- ible conformational transition gives rise to zigzag temperature de- pendence of the rate constant of irreversible thermodeactivation of enzymes, Eur: J. Biochem. 219, 219-230.

Wilcox, P. E. (1970) Chymotrypsinogens - chymotrypsins, Method Enzymol. 19, 64- 108.

Poole, R. K. & Bashford, C. L. (1987) Spectra, in Spectrophotome- try C? spectrojluorimetry (Harris, D. A. & Bashford, C. L., eds) pp. 23-48, IRL Press, Oxford.

Wada, A., Saito, Y. & Ohogushi, M. (1983) Multiphasic conforma- tion transition of globular protein under denaturing conditions, Biopolymers 22, 93-99.

Sadana, A. & Henley, J. P. (1986) Mechanistic analysis of complex enzyme deactivations. Influence of various parameters on series- type inactivations, Biotechnol. Bioeng. 28, 977-987.

Pace, C. N. (1990) Measuring and increasing protein stability, Trends Biotecknol. 8, 93-98.

Creighton, T. E. (1990) Protein folding, Biochem. J. 270, 1 - 16. Hess, G. P. (1971) Chymotrypsin, chemical properties and catalysis,

in The enzymes, 3rd edn (Boyer, P., ed.) vol. 111, pp. 213-248, Academic Press, New York.

Eng. 12,41-118.