Kinetics of N -glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by α-chymotrypsin in aqueous solutions of dodecyltrimethylammonium bromide

Abstract

The rate at which α-chymotrypsin (α-CT) catalyzes the hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) was measured in aqueous solutions containing dodecyltrimethylammonium bromide (DTAB) at concentrations both below and above the critical micelle concentration (CMC), as well as in solutions without any surfactant. Under all experimental conditions, the hydrolysis reaction followed the Michaelis–Menten kinetic model. The presence of the surfactant resulted in enhanced enzyme activity both below and above the CMC. When the reaction rate was plotted against the total substrate concentration, a maximum rate was observed near the CMC. A similar trend was observed when the enzyme was partially deactivated by the addition of 4 M urea. After correcting the observed reaction rates to account for the distribution of the substrate between the micelles and the bulk aqueous solution, the enzyme activity tended to remain relatively constant at surfactant concentrations above the CMC. This apparent constancy in activity resulted from a compensatory effect involving a decrease in both the catalytic rate constant (kcat) and the Michaelis constant (KM). The behavior of α-CT in the hydrolysis of GPNA in DTAB solutions contrasts with previously reported findings for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant. A possible explanation for the differing effects of the surfactant on the enzyme’s activity with these two substrates is proposed, considering the complex mechanism of the α-CT-mediated reaction, specifically in terms of different rate-limiting steps involved in the formation of the measured products.

Introduction

Since the pioneering work of Martinek and colleagues in 1977, micellar enzymology has emerged as a significant area of physicochemical research for investigating problems in molecular biology. This field has extensively explored enzyme-catalyzed reactions in reverse micellar solutions. In contrast, the study of biocatalysis in aqueous solutions containing surfactants has received considerably less attention.

Surfactants in aqueous solutions can influence the kinetics of enzymatic reactions at concentrations both below and above the critical micelle concentration (CMC). The interaction between the enzyme and the surfactant can lead to either a reduction or an enhancement of enzymatic activity, and these effects can be dependent on both the concentration and the specific characteristics of the surfactant.

In a previous study, it was demonstrated that dodecyltrimethylammonium bromide (DTAB) caused a decrease in the activity of α-chymotrypsin (α-CT) during the hydrolysis of 2-naphthyl acetate (2-NA). This inhibitory effect was observed only at surfactant concentrations above the critical micelle concentration and could not be simply attributed to a reduction in substrate availability due to its incorporation into the micellar pseudophase. Conversely, Alfani and co-workers have reported that α-CT exhibits superactivity in media rich in cetyltrialkylammonium bromide surfactants, using N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) as the substrate. This enhancement of activity occurred both below and above the CMC of the surfactants, and for cetyltrimethylammonium bromide (CTAB), the catalytic activity increased by nearly a factor of 13. The significant difference between these two sets of results could be due to variations in the length of the alkyl chain of the surfactants used and/or a strong influence of the substrate on how a given surfactant affects the catalytic activity of α-CT. To investigate this latter possibility, we conducted a study on the effect of DTAB on α-CT activity using GPNA as the substrate. The results obtained indicate that the response of the enzyme’s catalytic activity to the surfactant concentration is highly dependent on the specific substrate employed.

Experimental

N-glutaryl-L-phenylalanine p-nitroanilide (Sigma), α-chymotrypsin (Type II, from bovine pancreas; isoelectric point, pI = 8.8, Sigma), and urea (Scharlau) were used without further purification. Dodecyltrimethylammonium bromide (Sigma) was used as received and also after several recrystallizations from acetonitrile, yielding similar results. Tris(hydroxymethyl)aminomethane (Tris) was obtained from Aldrich. Pyrene (Molecular Probes) was used as received. Ultrapure water, obtained from a Modulab Type II equipment, was used to prepare all solutions. Absorption spectra and absorbance measurements were recorded using a Hewlett-Packard UV–visible 8453 spectrophotometer. Fluorescence measurements were performed using an Aminco-Bowman Series 2 luminescence spectrometer.

The rate of GPNA hydrolysis, catalyzed by α-CT, was measured in the absence of surfactant and in DTAB solutions at pH 7 (using a 10 mM Tris/HCl buffer). Some experiments were also conducted in the presence of 4 M urea to partially deactivate the enzyme. The reaction rate was monitored by recording the absorbance of the released p-nitroaniline (PNA) at 386 nm as a function of time. The molar absorption coefficient (ε) of PNA was taken as 12,500 M−1 cm−1, both in the buffer solution and in the presence of the surfactant. The reported values correspond to initial reaction rates (v0), which were determined from the slope of the PNA concentration versus time profiles at the beginning of the reaction.

The critical micelle concentration (CMC) of DTAB in the buffer solution and in the buffer solution containing 4 M urea was determined using pyrene as a fluorescence probe. This method relies on observing the effect of DTAB addition on the ratio of the fluorescence intensities of the first and third vibrational bands of pyrene (I1/I3). The onset of micelle formation is indicated by an abrupt change in the slope of the plot of the I1/I3 ratio against the DTAB concentration. The partition constant of GPNA between the micelles and the bulk aqueous medium (buffer or buffer plus 4 M urea) was determined from absorbance measurements using the procedure described by Sepulveda and colleagues.

Results and discussion

The formation of p-nitroaniline (PNA) exhibited a linear dependence on time during the initial 10 minutes of the reaction for all concentrations of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) tested. The initial reaction rate, denoted as v0, was determined from the slope of the [GPNA] versus time plots.

Representative plots illustrating the substrate saturation curves obtained in the absence of any surfactant and in the presence of varying concentrations of dodecyltrimethylammonium bromide (DTAB) are presented. Before analyzing these results, it is crucial to understand the effect of GPNA addition on the critical micelle concentration (CMC) of DTAB. These data are compiled. While a moderate decrease in the CMC is observed with increasing GPNA concentration, this effect is minimal under the conditions used in the kinetic experiments. Therefore, the results will be analyzed considering an average CMC value of 12.2 mM, irrespective of the GPNA concentration. The data reveal a noticeable increase in the reaction rate with increasing surfactant concentration, particularly at concentrations below the CMC. This trend is further emphasized by plotting v0, measured at a single GPNA concentration, as a function of DTAB concentration. This plot exhibits a pronounced bell-shaped curve with a maximum reaction rate occurring at a DTAB concentration near the surfactant CMC. This indicates significant superactivity below the CMC, followed by a decrease in the reaction rate at higher surfactant concentrations. To determine if this decrease is due to the reduced thermodynamic activity of the substrate resulting from its incorporation into the micellar pseudophase, we evaluated the partitioning of GPNA between the micellar pseudophase and the bulk aqueous medium.

According to the pseudophase model, when the mole fraction of GPNA in the micellar pseudophase is low, the partitioning of the substrate can be defined by the following relationship:

$K = \frac{[GPNA]_m}{[GPNA]_{free} [DTAB]_m}$

where $[GPNA]_m$ and $[GPNA]_{free}$ represent the analytical concentrations of GPNA incorporated into the micelles and remaining free in the external medium, respectively, and $[DTAB]_m$ is the concentration of micellized surfactant ($[DTAB]_{total} – CMC$). Absorbance measurements yielded a value of $K = (400 \pm 60) M^{-1}$ for this partitioning constant.

The values of v0 corrected for the concentration of free substrate, denoted as $(v_0)_{corr}$, are presented. These data indicate that, after this correction, the reaction rate changes only by a factor of 2 over a wide range of surfactant concentrations, from the CMC up to very high concentrations (100 mM).

The observed behavior of α-CT activity in DTAB solutions for the hydrolysis of GPNA is similar to that reported by Alfani and colleagues in cetyltrimethylammonium bromide (CTAB) solutions but is markedly different from our previous findings for the α-CT/DTAB system in the hydrolysis of 2-naphthyl acetate (2-NA). Specifically, the reaction rate measured in the present study increases nearly 13 times from pure buffer to approximately 10 mM DTAB, a value very close to the increase reported by Alfani and co-workers in CTAB solutions. In contrast, the α-CT activity remained independent of DTAB concentration up to the surfactant CMC when 2-NA was used as the substrate. The current results suggest that this significant difference arises from the fact that the enzyme’s behavior is highly sensitive to the characteristics of the substrate, such as its shape and hydrophobicity. It is important to note that, above the CMC, superactivity is observed in the present work after correcting for substrate partitioning, whereas a loss of activity was reported for the hydrolysis of 2-NA under the same conditions. This further emphasizes the dependence of the DTAB effect on the substrate’s properties.

To determine whether the significant superactivity observed with GPNA is due to an increase in the catalytic rate constant (kcat) and/or a decrease in the Michaelis constant (KM), the kinetic data were analyzed using the Lineweaver–Burk plot. The resulting plots were linear. The derived values of kcat and KM (corrected for free substrate concentration) are shown. These data indicate that, below the CMC, kcat increases and KM decreases as the surfactant concentration increases. Both of these factors contribute to an increase in the enzyme’s efficiency, with the surfactant having a more pronounced effect on kcat. In this surfactant concentration range, we can conclude that both the binding of the substrate to the enzyme and the enzyme’s turnover rate are enhanced by the presence of the surfactant. The results obtained above the CMC are less straightforward. The minimal change in $(v_0)_{corr}$ with increasing DTAB concentration above the CMC appears to be a result of a compensation between a decrease in kcat and a decrease in KM. It was not possible to obtain reliable kcat and KM values at high surfactant concentrations (above 20 mM) due to the large errors associated with their estimation.

The v0 versus DTAB concentration profile was also determined in the presence of 4 M urea at a GPNA concentration of 0.1 mM. The resulting plot also exhibited a bell-shaped behavior with a maximum reaction rate observed at a DTAB concentration near the CMC in the 4 M urea solution. Furthermore, when the reaction rates, calculated based on the analytical concentration of GPNA, were corrected for the free substrate concentration using a partition constant $K_{(4 M urea)} = (65 \pm 10) M^{-1}$, the enzyme’s activity tended to remain constant at DTAB concentrations above the CMC, similar to the case where the reaction occurred in the absence of urea. Interestingly, although the maximum rate in the presence of urea was shifted to higher DTAB concentrations, at 20–30 mM DTAB, the reaction rate in 4 M urea was similar to or even higher than that observed without urea. This seemingly anomalous result can be explained by considering the different partition constants in each medium, which lead to different degrees of reduction in the substrate’s thermodynamic activity due to its incorporation into the micelles.

The results obtained in this study indicate that the different behaviors observed for the α-CT-catalyzed hydrolysis of GPNA and 2-NA in aqueous solutions of alkyltrimethylammonium bromide surfactants are not due to the length of the surfactant’s alkyl chain. Instead, the enzyme’s behavior is strongly influenced by the specific substrate employed. In this context, it is interesting to note that Eremeev and colleagues reported similar findings for the α-CT-catalyzed hydrolysis of different substrates in mixtures of homogeneous solvents, where bell-shaped or continuously decreasing v0 rates were observed depending on the substrate considered.

A possible explanation for the different effects of DTAB on the hydrolysis of GPNA and 2-NA by α-CT can be advanced by considering the complexity of the α-CT-mediated hydrolysis mechanism.