Kinetics of 2-Naphthyl Acetate Hydrolysis Catalyzed by α

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Kinetics of 2-Naphthyl Acetate Hydrolysis Catalyzed by r-Chymotrypsin in Aqueous Solutions of Dodecyltrimethylammonium Bromide Elsa Abuin,* Eduardo Lissi, and Roxanna Duarte Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Casilla 40-Correo 33, Santiago, Chile Received February 6, 2003. In Final Form: April 23, 2003 The rate of hydrolysis of 2-naphthyl acetate catalyzed by R-chymotrypsin has been measured in aqueous solutions of dodecyltrimethylammonium bromide at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction takes place following a Michaelis-Menten mechanism. The presence of the surfactant, at concentrations above its critical micellar concentration, increases the value of the Michaelis constant, Km, without significant changes in the catalytic rate constant, kcat. The increase in Km is larger than that expected from the incorporation of the substrate to the micellar pseudophase, indicating that the interaction between the enzyme and micellelike aggregates alters the formation of the enzyme-substrate complexes. This can be related to a partial unfolding of the enzyme, as is suggested by changes in its intrinsic fluorescence.

Introduction Since 1977, after the pioneer work of Martinek et al.,1 micellar enzymology has arisen as a new physicochemical line of research to approach problems in molecular biology. The field has mainly covered the study of reactions catalyzed by enzymes in reverse micellar solutions,2-17 the effect of aqueous solutions of surfactants upon the kinetics of enzymatic reactions being less explored.12,18-23 In principle, surfactants in aqueous solutions could * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 56-2-6812108. (1) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Berezin, I. V. Dokl. Acad. Nauk SSSR 1977, 236, 920. (2) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (3) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (4) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C. J. Chem. Soc., Faraday Trans. 1 1985, 81, 2667. (5) Bru, R.; Sa´nchez-Ferrer, A.; Garcı´a-Carmona, F. Biochem. J. 1989, 259, 355. (6) Verhaert, R. M. D.; Hilhorst, R.; Vermue¨, M.; Schaafsma, T. J.; Veeger, C. Eur. J. Biochem. 1990, 187, 59. (7) Bru, R.; Sa´nchez-Ferrer, A.; Garcı´a-Carmona, F. Biochem. J. 1990, 268, 679. (8) Bru, R.; Walde, P. Eur. J. Biochem. 1991, 199, 95. (9) Sarcar, S.; Jain, T. K.; Maitra, A. Biotechnol. Bioeng. 1992, 39, 474. (10) Miyake, Y.; Owari, T.; Matsuura, K.; Teramoto, M. J. Chem. Soc., Faraday Trans. 1993, 89, 1993. (11) Stamatis, H.; Xenakis, A.; Menge, U.; Kolisis, F. Biotechnol. Bioeng. 1993, 42, 931. (12) Miyake, Y.; Owari, T.; Ishiga, F.; Teramoto, M. J. Chem. Soc., Faraday Trans. 1994, 90, 979. (13) Miyake, Y. Colloid Surf., A 1996, 109, 255. (14) Das, P. K.; Chaudhuri, A. Langmuir 2000, 16, 76. (15) Carvalho, C. M. L.; Aires-Barros, M. R.; Cabral, J. M. S. Langmuir 2000, 16, 3082. (16) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (17) Aguilar, L. F.; Abuin, E.; Lissi, E. Arch. Biochem. Biophys. 2001, 388, 231. (18) Martinek, K.; Levashov, A. V.; Klyachko, N.; Klmelnitski, Y. L.; Berezin, I. V. Eur. J. Biochem. 1986, 155, 453. (19) Spetri, N.; Alfani, F.; Cantarella, M.; D’Amico, F.; Germani, R.; Savelli, G. J. Mol. Catal. 1999, 6, 99. (20) Viparelli, P.; Alfani, F.; Cantarella, M. Biochem. J. 1999, 344, 765. (21) Alfani, F.; Cantarella, M.; Spreti, N.; Germani, R.; Savelli, G. Appl. Biochem. Biotechnol. 2000, 88, 1. (22) Viparelli, P.; Alfani, F.; Cantarella, M. J. Mol. Catal. B: Enzym. 2001, 15, 1.

affect the kinetic behavior of enzymatic reactions below or above the critical micelle concentration (cmc) as a consequence of two factors, namely: (i) an interaction between the enzyme and the surfactant, which can comprise the free surfactant and micelles, leading to conformational changes that can modify the catalytic rate constant and enzyme-substrate binding constant; and (ii) the partitioning of the substrate between the micelles and the external solvent. R-Chymotrypsin is a water-soluble enzyme that catalyzes the hydrolysis of peptidic bonds in proteins, being also able to act upon simple amides3,19-22 and esters.12 The activity of the enzyme is modified by the presence of lipid/water interfaces, and extensive studies have been reported regarding the characteristics of 2-naphthyl acetate (2-NA) hydrolysis in AOT [sodium bis(2-ethylhexyl)sulfosuccinate] reverse micelles.12,17 Nevertheless, limited data have been reported regarding the effect of this anionic surfactant upon the activity of the enzyme in the hydrolysis of 2-NA in an aqueous solution.12 On the other hand, Alfani and co-workers have carried out a comprehensive study of R-chymotrypsin-catalyzed hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide in the presence of cetyltrialkylammonium bromides.19-22 In the present work, we report the results of a study on the effect of a cationic surfactant, dodecyltrimethylammonium bromide (DTAB), upon the rate of hydrolysis of 2-NA catalyzed by R-chymotrypsin in an aqueous solution. The results obtained are discussed in terms of the effect elicited by the surfactant upon the catalytic rate constant, the Michaelis constant, and the partitioning of the substrate. The kinetic aspects of the reaction are complemented with information obtained from the analysis of the fluorescence behavior of the tryptophan residues of the enzyme in the presence of the surfactant. Experimental Section DTAB (Sigma), 2-NA (Sigma), R-chymotrypsin (Type II, from bovine pancreas, pI ) 8.8 Sigma), and urea (Scharlau) were used as were received. Ultrapure water obtained from Modulab Type (23) Berg, O. G.; Rogers, J.; Yu, B.-Z.; Yao, J.; Romsted, L. S.; Jain, M. K. Biochemistry 1997, 36, 14512.

10.1021/la030050s CCC: $25.00 © 2003 American Chemical Society Published on Web 05/23/2003

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Figure 1. Effect of the DTAB concentration on the relationship between the initial rate of reaction and the analytical concentration of 2-NA: [R-chymotrypsin] ) 1 mg mL-1. (b) In the absence of DTAB, ([) [DTAB] ) 5 mM, (9) [DTAB] ) 10 mM, (2) [DTAB] ) 20 mM, and (1) [DTAB] ) 50 mM. II equipment was employed to prepare all the solutions. The absorption spectra and absorbances were recorded in a Hewlett Packard UV-visible 8453 spectrophotometer. The fluorescence spectra were registered using an Aminco-Bowman Series 2 luminescence spectrometer. The rate of 2-NA hydrolysis, catalyzed by R-chymotrypsin, was measured in an aqueous solution and in DTAB solutions at pH ) 7 (10 mM Tris/HCl buffer). Because the isoelectric point of the enzyme is 8.8, it has a neat positive charge under the conditions employed in this work. The reaction was followed by registering the absorbance of 2-naphthol (2-N) at 330 nm ( ) 1.53 × 103 M-1 cm-1) as a function of time. Initial rates, V0, were determined from the slope of the 2-N concentration versus time profiles measured at time t f 0. In the fluorescence experiments, the enzyme was excited at λ ) 295 nm to selectively isolate the fluorescence arising from the tryptophan residues.24 The emission spectra were registered in the 310-400-nm range.

Results and Discussion A. Reaction in the Aqueous Phase in the Absence of the Surfactant. The initial rate of 2-NA hydrolysis, V0, was calculated from the slope of the 2-N concentration versus time plots at t f 0. Due to the low solubility of 2-NA in the buffer (ca. 0.3 mM), the dependence of V0 on the 2-NA concentration can be determined over only a limited range of 2-NA concentrations (up to 0.2 mM). The V0 versus 2-NA concentration profile obtained is included in Figure 1. The data plotted according to the LineweaverBurk treatment, eq 1,

[E]/V0 ) 1/kcat + Km/kcat[2-NA]-1

(1)

where [E] is the enzyme concentration, kcat is the catalytic rate constant, and Km is the Michaelis constant, are shown in Figure 2. The linearity of the plot indicates that, under the conditions employed, the Michaelis-Menten mechanism applies. (24) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1986; chapter 11.

Figure 2. Lineweaver-Burk plot for the R-chymotrypsincatalyzed hydrolysis of 2-NA: (b) in the absence of DTAB and (2) [DTAB] ) 20 mM.

B. Reaction in the Presence of the Surfactant. Representative results obtained for V0 versus [2-NA]analyt (analytical concentration of 2-NA) profiles in the presence of surfactant are included in Figure 1. The data cannot be associated with a simple salt effect25 because the reaction rates measured in the presence of 2 M tetramethyltrimethylammonium bromide were the same as those determined in pure buffer. A meaningful analysis of these profiles shown in Figure 1 requires a knowledge of the cmc of DTAB in the buffer employed. The cmc was determined following the effect elicited by the DTAB addition upon the fluorescence of 2-NA. The onset of micellization was evidenced by an abrupt change in the slope of a plot of I0/I values (I0 and I being the fluorescence intensities in the absence and presence of DTAB, respectively) versus [DTAB], (Figure 3b). The increase of I0/I values with [DTAB] after the cmc is associated with the incorporation of 2-NA to the micelles because the fluorescence of 2-NA in the aggregates is strongly quenched as a result of the high local Brconcentration at the micellar surface.26 From the data shown in Figure 3b, a cmc ) 14 mM was obtained, a value very close to that reported for the cmc of DTAB in pure water.27 The results shown in Figure 1 indicate that, at DTAB concentrations below the cmc (5 and 10 mM), V0 versus [2-NA]analyt profiles are very similar to that in the absence of surfactant. On the other hand, in the presence of micelles, the V0 versus [2-NA]analyt profiles are strongly dependent on the DTAB concentration, with a strong decrease in V0 values when the surfactant concentration increases. The data obtained at [DTAB] ) 20 mM, plotted according to eq 1, are included in Figure 2. The linearity of the plot indicates that, in the presence of micelles, a Michaelis-Menten mechanism still applies. From the convergence to the same intercept (eq 1) of the data obtained in buffer and 20 mM DTAB, it can be concluded that the presence of the micelles does not significantly (25) Cohn, M.; Kesslinger, S. J. Biol. Chem. 1960, 235, 1365. (26) Abuin, E.; Lissi, E.; Quina, F.; Sepulveda, L. J. Phys. Chem. 1984, 88, 81. (27) Abuin, E.; Scaiano, J. C. J. Am. Chem. Soc. 1984, 106, 6274.

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Figure 4. Data of Figure 3 after the surfactant cmc plotted according to eq 3.

Rearrangement of eq 2 leads to eq 3:

fb/ffree ) K[DTAB]m

(3)

The value of K was derived from the data shown in Figure 3a, considering that eq 4 applies for fb

fb ) (I0/I - 1)/[(I0/I)∞ - 1]

Figure 3. (a) Effect of DTAB addition upon the relative fluorescence intensity, I0/I, of 2-NA. (b) Plot of the data at low DTAB concentration used to obtain the surfactant cmc. Excitation wavelength ) 290 nm and emission wavelength ) 335 nm.

affect the catalytic rate constant. A common value of kcat ) 8.8 × 10-6 mol g-1 s-1 can be derived from the plots shown in Figure 2. On the contrary, the slope of the line obtained in the presence of micelles is much larger than that corresponding to the reaction in buffer. Because the reactions taking place under both conditions have the same kcat, the effect of micelles on the slope (as well as the smaller values of V0 obtained in the presence of micelles, Figure 1) can be due to partitioning of the substrate between the micelles and the external medium or the effect of the micelles upon the Michaelis constant. According to the pseudophase model,28 when the mole fraction of 2-NA in the micellar pseudophase is low, the partitioning of the substrate can be defined through eq 2:

K ) fb/(ffree[DTAB]m)

(2)

where K is the partition constant, fb is the fraction of 2-NA bound to the micelles (fb ) [2-NA]b/[2-NA]analyt), ffree is the fraction of 2-NA remaining in the aqueous pseudophase (ffree ) [2-NA]free/[2-NA]analyt), and [DTAB]m is the concentration of micellized surfactant ([DTAB]analyt - cmc). (28) Sepulveda, L.; Lissi, E.; Quina, F. Adv. Colloid Interface Sci. 1986, 25, 1.

(4)

where I0/I corresponds to a given value after the cmc and (I0/I)∞ - 1 corresponds to 100% of the binding. The plot obtained is shown in Figure 4. From the slope of this plot, a K value of 180 ( 10 M was obtained. The lack of curvature in the V0 versus [2-NA]analyt profile for the reaction carried out at [DTAB] ) 50 mM (Figure 1) precludes the determination of the apparent Michaelis constant, (Km)app, at this surfactant concentration. Nevertheless, from the slope/intercept ratio of the lines shown in Figure 2, values of (Km)buffer ) 5.8 × 10-5 M and (Km)app ) 3.2 × 10-4 M can be derived for the reaction carried out in buffer and [DTAB] ) 20 mM, respectively. The increase in (Km)app (determined in terms of the analytical concentration of 2-NA) in the presence of micelles can be due to an intrinsic micellar effect upon this parameter or to the decreased activity of the substrate resulting from its incorporation to the micellar pseudophase. With the aim to differentiate these possibilities, the value of (Km)app obtained at [DTAB] ) 20 mM was corrected to take into account the partitioning of the substrate. To this effect, (Km)app was multiplied by the fraction of 2-NA remaining in the aqueous pseudophase according to eq 5, leading to a (Km)corr (corrected for substrate partitioning) value

(Km)corr ) (Km)app/(1 + K[DTAB]m) ) (Km)app ffree (5) From eq 5, (Km)corr ) 1.5 × 10-4 M was obtained at [DTAB] ) 20 mM total surfactant concentration, leading to (Km)buffer/(Km)corr ) 0.39. Another way to visualize the micellar effect upon the reaction kinetics that permits one to include the data obtained at the two micellized surfactant concentrations considered is based on the analysis of the dependence of the initial slope (R) of the plots of Figure 1 on the fraction of 2-NA remaining in the aqueous pseudophase, ffree. If the micellar effect upon the reaction rate was exclusively due to the sequestering of the substrate by the micelles,

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Table 1. Dependence of Z on ffreea [DTAB]total (mM)

ffree

Z

[DTAB]total (mM)

ffree

Z

0 5 10

1 1 1

1 =1 =1

20 50

0.48 0.13

0.38 0.28

a

See text for definitions.

R values divided by ffree must be independent of ffree. In other terms, taking the R value in the buffer as the reference (ffree ) 1), a parameter Z defined in eq 6 can be considered to analyze the effect of the surfactant upon the enzyme activity.

Z ) Rm/(Rbuffer ffree)

(6)

The values of Z obtained from eq 6 are shown in Table 1. The last column of Table 1 gives the loss of activity associated with substrate partitioning. The value for [DTAB] ) 20 mM is in good agreement with the analysis made in terms of the slopes of Figure 2. Nevertheless, it is seen that the loss of activity is not independent of ffree, being higher for the reaction carried out at [DTAB] ) 50 mM. This indicates that not only substrate partitioning but also a true micellar effect upon the Michaelis constant have to be considered. Similar conclusions have been obtained regarding the effect of AOT in the aqueous phase on the hydrolysis of 2-NA,12 where a decrease in the enzyme activity has been related to an increase in the Michaelis constant as a result of the instability of the enzymesubstrate complex, elicited by the adsorption of AOT molecules on the enzyme. An effect of the surfactant on the Michaelis constant at concentrations above the cmc suggests that the enzyme interacts with the micelles (or that micellelike aggregates are formed on the enzyme) in such a way that its native conformation changes to another one in which the enzyme-substrate association is reduced. To obtain some information on possible enzyme conformational changes, we have registered the fluorescence spectra of R-chymotrypsin in pure buffer, in the presence of the surfactant and urea. The results obtained are shown in Table 2. In

Table 2. Effect of DTAB or Urea Addition upon the Fluorescence Spectra of r-Chymotrypsin additive

wavelength at the maximum (nm)

none DTAB (10 mM) DTAB (20 mM) DTAB (50 mM) urea (8 M)

334 334 337 338 356

line with the kinetic experiments, the spectra are unmodified at DTAB concentrations below the cmc. In the presence of micelles, a moderate shift to the red of the wavelength of maximum fluorescence takes place. This shift is considerably smaller than that associated with the total unfolding of the enzyme, as was measured in the presence of 8 M urea. From these results, it can be concluded that the micellar effect upon Km is due to a weaker substrate association to the partially unfolded enzyme. The results obtained in the present work regarding the effect of DTAB micelles on the enzyme-catalyzed hydrolysis of 2-NA bear noticeable differences with those reported for the activity of the enzyme in the hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide in cetyltrialkylammonium bromide-rich media.20,21 In these systems, when several surfactants of different headgroups were employed, it was found that there was either superactivity below the cmc21 and superactivity (cetyltributylammonium bromide) or loss of activity (cetyltrimethylammonium bromide) in the presence of micelles.20 The differences with the present data (no effect below the cmc and decreased activity above it), obtained for 2-NA in the presence of DTAB, emphasizes the sensitivity of the enzyme behavior to the substrate and surfactant alkyl chain length. Further experiments are being carried out to evaluate the relevance of each one of these factors. Acknowledgment. Financial support of this work by DICYT (USACH) and FONDECYT (Project No. 1010148) is acknowledged. LA030050S