Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1100-1104, February 1992 Biochemistry

Enzymatic catalysis and dynamics in low-water environments RHETT AFFLECK*, ZU-FENG XUt, VALERIE SUZAWA*, KATHLEEN FOCHTt, DOUGLAS S. CLARK*1, AND JONATHAN S. DORDICKtt *Department of Chemical Engineering, University of California, Berkeley, CA 94720; and tDepartment of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242

Communicated by T. Kent Kirk, October 28, 1991 (received for review July 28, 1991)

Enzymes suspended in organic solvents repABSTRACT resent a versatile system for studying the involvement of water in enzyme structure and function. Addition of less than 1% (vol/vol) water to tetrahydrofuran containing 1 M 1-propanol leads to a substantial increase in the transsterification activity of subtilisin Carlsberg (from Bacilus licheniformis) that correlates with a sharp increase in the active-site polarity and a 90% decrease in the rotational correlation time (i.e., increase in mobility) of a nitroxide spin label within the active site. Water in excess of 1% has little additional effect on active-site polarity and coincides with a further increase in spin-label mobility, yet the transesterification activity decreases dramatically. Thus, transesterification activity increases and then decreases with increasing enzyme hydration and flexibility (which are presumably coupled through dielectric screening), suggesting that the conformation of partiaily hydrated subtilisin is diflerent from that of the nearly dry enzyme-4.e., enzyme containing less than 9% (wt/wt) water. The physicochemical properties of enzymes depend largely on the direct or indirect role of water in various noncovalent interactions including solvation of ionic groups and dipoles, hydrogen bonding, and hydrophobic interactions (1, 2). Most studies aimed at elucidating the role of water in enzyme structure and function have utilized hydrated protein powders or films (3-10). For example, studies of lysozyme powders equilibrated with water in air have shown that catalysis occurs at a hydration level (ca. 0.2 g of water per g of enzyme) well below monolayer water coverage (10). NMR analysis of the hydration process indicates that the onset of catalytic activity is a direct consequence of an increase in lysozyme's conformational flexibility (3, 11). It has been suggested that this increased flexibility may be due, in part, to the reduced interaction of charged and/or polar amino acid residues within the enzyme molecule caused by water's ability to effect dielectric screening (12). Thus, water may act both as a plasticizer to increase the structural flexibility of an enzyme molecule (3) and as a solvent to dielectrically screen unfavorable interactions between charged and/or polar residues within the protein molecule (3, 12). However, the use of enzyme powders complicates the measurement of enzyme activity, particularly at low catalytic activities. Hence, the molecular basis for the induction of enzyme function by water remains unclear. An alternative to hydrated powders is to use enzymes in partially hydrated organic solvents. Enzymes are well known to function in nonaqueous media (13-15). As in hydrated powders, enzyme function in nonaqueous media is strongly dependent upon the water content of the enzyme (16, 17). However, the role of water in molecular events at the enzymic active site leading to catalysis in low-water environments is not well understood. In this work, we have

probed the active site structure and dynamics of subtilisin in nonaqueous media and have correlated observed structural/ chemical changes in the environment of an active-site spin label with both enzyme hydration and catalytic function. Subtilisin Carlsberg was chosen for these studies for several reasons: (i) the enzyme is cofactor-independent and, hence, cofactor-enzyme interactions need not be considered; (ii) the three-dimensional structure of the enzyme is known to 1.8 A (18); and (iii) subtilisin is active in a wide variety of organic solvents and is catalytically active even with very little bound-water [ca. 9%o (wt/wt)] (16).

MATERIALS AND METHODS Materials. Subtilisin Carlsberg (from Bacillus licheniformis) was obtained from Sigma. The enzyme was activated prior to use in organic media by lyophilization from aqueous 20 mM phosphate buffer (pH 7.8). In this manner, the ionogenic groups of the enzyme are retained in their catalytically correct form in nonaqueous media (13-15). Subtilisin BPN' (from Bacillus amyloliquefaciens) was supplied by James Wells, Genentech. The spin label, 4-(ethoxyfluorophosphinyloxy)-TEMPO (TEMPO = 2,2,6,6-tetramethyl-1piperidinyloxy, free radical), was obtained from Aldrich. All other chemicals and solvents used in this work were of the highest grade commercially available. All solvents were twice distilled and stored over molecular sieves (3 A, Linde) at least 24 hr prior to use. Kinetic Studies. The catalytic efficiencies of subtilisin Carlsberg were determined in tetrahydrofuran containing different concentrations of water. The reaction studied was the transesterification of N-acetyl-L-phenylalanine 2-chloroethyl ester (NAPCE) with 1-propanol. In a typical experiment, 2 mg of subtilisin powder was added to 2 ml of tetrahydrofuran containing 10-50 mM NAPCE and 1 M 1-propanol. The enzyme suspension was mixed vigorously in a Vortex for 10 sec, and water was then added (ranging from no added water to a total of 140 1. of water), and the suspension was mixed for 10 sec and sonicated for an additional 30 sec. No evidence of enzyme interacting with the glass vial was obtained. The reaction mixture was shaken at 250 rpm at 300C. Initial rate measurements were performed over a 4- to 8-hr period, during which time no loss in enzyme activity occurred. The reaction mixtures were analyzed by gas chromatography (GC) and high-performance liquid chromatography (HPLC). The former method measured the formation of the N-acetyl-L-phenylalanine propyl ester (NAPPE) transesterification product by using an HP-1, 530-,um fused silica capillary column (Hewlett-Packard) of 25-m length and 0.33-,um inside diameter with a N2 carrier gas flow rate of 0.5 ml/min, 250°C injection and detection temAbbreviations: TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy free radical; NAPCE, N-acetyl-L-phenylalanine 2-chloroethyl ester; NAPPE, N-acetyl-L-phenylalanine propyl ester; ST-ESR, saturation-transfer electron spin resonance. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.


Biochemistry: Affleck et A peratures, and an initial column temperature of 1800C for 1 min, which thereafter increased to 250'C at a rate of 350C/ min. Hydrolysis of NAPCE in solutions with added water was measured by HPLC by using a ABondapak C18 reversephase column with a 24% acetonitrile solution in 10 mM ammonium phosphate buffer (pH 2.3) as eluant at a flow rate of 1.5 ml/min. Detection of the NAPPE product was performed at 280 nm with a photodiode array detector (model 990, Waters). The kinetic parameter Vma.,/Km was determined by nonlinear Michaelis-Menten fitting of the rate data with a nonlinear regression algorithm (19). The kinetic data were converted into intrinsic catalytic efficiencies (kcat/Km) by normalizing Vmj/Km by the concentration of active enzyme used. This was done via activesite titration using the active-site reversible inhibitor N-transcinnamoylimidazole as described by Zaks and Klibanov (16). In aqueous buffer, all enzymic active sites were accessible to the solvent, and 63% of the enzyme was active. In dry tetrahydrofuran containing 1 M 1-propanol, roughly 15 ± 4% of the subtilisin's active sites were accessible to the organic solvent within 48 hr, at which point equilibrium was reached. This fraction did not change significantly upon addition of water up to 20 jkl/ml. Above this water concentration, the fraction of active sites exposed to the solvent increased dramatically; when added water reached 70 pu/ml, all possible active sites were accessible to the solvent. Determination of Enzyme-Bound Water. The enzymebound water was measured as follows (16): 1 mg of subtilisin per ml was suspended in tetrahydrofuran containing a specific concentration of water. The suspension was shaken at 250 rpm at 30'C for 10 min, after which the mixture was centrifuged and the supernatant was decanted. The water content of the supernatant was then determined by Karl Fischer coulometric titration. The remaining solid material with entrained solvent (the water content of the entrained solution was assumed to be equivalent to the decanted supernatant) was then weighed. The solvent volume was

Proc. Natl. Acad. Sci. USA 89 (1992)


calculated from the difference between the measured weight of the total sample and the weight of the enzyme initially added. The water content ofthe entire mixture was then determined by Karl Fischer titration. The amount of water bound to the subtilisin was the difference between the measured water and the water calculated to be in the entrained solvent. Electron Spin Resonance (ESR) Studies. Subtilisin was spin-labeled with 4-(ethoxyfluorophosphinyloxy3-TEMPO as described by Morrisett and Broomfield (20). The spin-labeled enzyme was suspended in organic solvent solutions containing the water concentrations indicated and transferred via a syringe to glass capillary tubes for ESR measurements. Conventional spectra were recorded at room temperature on a Bruker ER200D-SRC ESR spectrometer with a microwave power of 12.6 mW, a modulation amplitude of 1.0 G (1 G = 0.1 mT), and a scan range of 150 G._-To obtain saturation transfer ESR (ST-ESR) spectra, out-of-phase second harmonic absorption spectra were phased using the "self-null" method (21). The modulation frequency was 50 kHz, the modulation amplitude was 5 G, and the microwave power was 350 mW. For active-site polarity calculations, the Ao values were determined from computer simulations of ESR spectra recorded at 120 K. Rigid-limit computer simulations using standard first-order transition energies (22) were performed with a program obtained from J. H. Freed and co-workers (Dept. of Chemistry, Cornell Univ.). Best-fit simulations were obtained by allowing components of the A and g tensors to vary in order to maximize the agreement between experimental and calculated spectra. For the simulations, the x axis was taken as being along the N-O bond, the z axis along the


'° 0.6* 4

0.4 0










a) 0

40 Water added, ,l/ml FIG. 1. Water adsorption isotherm for subtilisin Carlsberg in tetrahydrofuran. The conditions were subtilisin at 1 mg/ml suspended in tetrahydrofuran containing 1 M 1-propanol with different concentrations of added water. The data points shown represent triplicate measurements with standard deviations of less than 5% of the measured values. Shaking for 60 min had no effect on the equilibrium of water binding onto the enzyme.


40 Water added, ,ul/ml


FIG. 2. Catalytic efficiencies of subtilisin Carlsberg in tetrahydrofuran with different water contents. The rate of NAPPE formation from NAPCE was determined by GC. Hydrolysis was not observed (as determined by the lack of appearance of N-acetyl-Lphenylalanine on reverse-phase HPLC) below 30 1d of added water per ml of solvent. While the catalytic efficiencies of subtilisin are ca. 3 orders of magnitude below values in aqueous solutions, catalysis in an organic medium is a result of active-site chemistry as shown in two control experiments: (i) subtilisin preinactivated with phenylmethanesulfonyl fluoride was nearly completely inactive in tetrahydrofuran (at least 4 orders of magnitude less active than the pHadjusted enzyme-the sensitivity limit for detection of the ester product by GC); and (ii) the activity of an active-site mutant S221A [whereby alanine replaces serine at the active site (position 221) of

the subtilisin] for the related enzyme from B. amyloliquefaciens (subtilisin BPN') is also at least 4 orders of magnitude less active than the pH-adjusted enzyme.


Biochemistry: Affleck et al.

2p ir orbital of the nitrogen, and the y axis perpendicular to the other two. RESULTS AND DISCUSSION In the absence of added water in tetrahydrofuran [

Enzymatic catalysis and dynamics in low-water environments.

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in enzyme structure and function. Addition of...
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