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
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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, 250C 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.

1100

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)

1101

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

0.8-

' 0.6*
4

30-

0.4 0

0"O

0.2

E20c

a)

0.0
0

0
a)
0

20

40 Water added, ,ul/ml

80

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.

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.

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Water added

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

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 [<0.01% (vol/vol) water] containing 1 M 1-propanol, the enzyme contains about 9%6 (wt/wt) water. Under these conditions, subtilisin exhibits low but measurable transesterification activity with NAPCE and 1-propanol (kcat/Km = 0.15 M-1ls'1). An increase in the water content of the tetrahydrofuran causes an increase in the level of enzyme hydration, up to ca. 35% (wt/wt; the calculated monolayer coverage based on the assumption that subtilisin is a spherical protein of Mr 27,500) in tetrahydrofuran containing 70 ,Al of water per ml. The enzymic hydration level follows a Langmuir-type adsorption isotherm (Fig. 1). The level of enzyme hydration has a significant effect on the catalytic activity of subtilisin in tetrahydrofuran (Fig. 2). The addition of 2 ,l of water per ml of tetrahydrofuran doubles the catalytic efficiency of transesterification, while addition of 5 pl of water per ml increases the catalytic efficiency more than 6-fold. This increase in catalytic efficiency is not due to an increase in the fraction of enzyme active sites exposed to the organic solvent, as this fraction does not significantly change upon addition of water up to 20 dul/ml. However, further addition of water causes a dramatic inactivation of subtilisin toward the ester substrate. Specifically, a loss in catalytic efficiency by a factor of 7 results from an increase in water from 5 pl to 20 Al per ml of tetrahydrofuran. This drop in activity is not due to competing hydrolysis in the presence of higher concentrations of water, as hydrolysis remains suppressed below a water content of 30 ,l per ml of tetrahydrofuran. To investigate whether the initial increase in catalytic efficiency at low water content in tetrahydrofuran may be due to an increase in the flexibility of the enzyme, the mobility of a nitroxide spin label bound to Ser-221 (the active-site nucleophile) was examined over a wide range of water concentrations. Shown in Fig. 3 are conventional and saturation transfer ESR spectra of subtilisin spin-labeled at its active-site serine with a fluorophosphonate spin label. The spectrum in tetrahydrofuran containing 70 AlI of added water per ml is similar to that of subtilisin dissolved in aqueous buffer and is characteristic of intermediate spin-label mobility. Spectral simulations (23) indicate that the average rotational correlation time of the spin label in this case is about 3 nsec. However, as the concentration of added water decreases, the spectrum broadens, reflecting a longer rotational correlation time and reduced mobility of the spin label. In the range of 0-5 pl of added water per ml of tetrahydrofuran, the conventional spectrum is nearly identical to that of the rigid, dry enzyme (free enzyme powder). Thus, the spectra in this range of added water represent the rigid limit of conventional ESR and are not very sensitive in shape to motions slower than about 100 nsec. In contrast, ST-ESR, which is sensitive to much slower motions than conventional ESR (21); showed a substantial increase in spin-label mobility with added water from 0 to 20 Al/ml. Hence, the increased catalytic efficiency of subtilisin in the presence of 5 pl of water per ml of tetrahydrofuran is accompanied by an increase in the motion of the spin label and, presumably, by greater flexibility of the enzyme's active site. Moreover, the decrease in catalytic activity of subtilisin when the water content is above 5 pA/ml coincides with a further increase in the spin-label's motion. In addition to the nearby dynamics, the polarity of the spin label's environment can be determined by measuring one of the four splitting constants, A0, A=, Ayy, or AZZ, for the N-O group of the spin label. Ax, Ayy, and Azz comprise the diagonal A matrix of the Hamiltonian written in the nitroxide

Water added

(PI/ml)

(MI/ml)
0
2

Water on enzyme (wt %)

9

20

19

35

26 33

lOG

lOG

FIG. 3. Saturation transfer (Left) and conventional (Right) ESR spectra of subtilisin spin-labeled at Ser-221 with 4-(ethoxyfluorophosphinyloxy)-TEMPO. The spin labeling stoichiometry, determined by twice integrating the ESR spectrum, was about 0.8 spinlabel molecules per enzyme. In separate experiments conducted with free spin label, the spectral lineshapes were independent of the water concentration. Thus, the differences illustrated above are specific to enzyme-bound spin label. Rotational correlation times were estimated from the ST-ESR spectra by comparing the central region of each spectrum to the calibration curve prepared by Thomas et al. (21) for spin-labeled hemoglobin. The calibration curve was shifted to obtain agreement between the rotational correlation times determined from the ST-ESR spectrum and a simulation of the conventional spectrum for added water at 20 ,ul/ml of solvent. The estimated rotational correlation times are 1500 nsec at 0 added water (based on

ST-ESR), 750 nsec at 2 A.l/ml (based on ST-ESR), 140 nsec at 5 ,uI/ml (based on ST-ESR), ca. 17 nsec at 20 jl/ml (based on spectral simulations), and ca. 3 nsec at 70 ld/ml (based on spectral simulations). The conventional spectra at water concentrations more than

5 juI/ml of solvent contain two components, which reduces the accuracy of the simulations.

coordinate system; AO is the average of the three. For a spin label undergoing anisotropic reorientation, none of these parameters can be measured directly at room temperature because of partial motional averaging of the electron-nuclear dipolar interaction. However, they can be determined from computer simulations of experimental ESR spectra recorded at very low temperature-i.e., in the absence of spin-label motion. Variations in the local polarity under different conditions can then be related to relative changes in the splitting constants. Although the environment of the spin label at low temperatures may differ somewhat from that at room temperature, differences in the active-site polarity of samples measured at low temperatures should still reflect differences between the same samples at room temperature.

Biochemistry: Affleck et A
A 15.75

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

1103

15.70

15.65
15.60
U) 15.55
Ca

CD 15.50 15.45
15.40

15.35 15.30 15.25

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IK
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30

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40

50

60

70

80

Water added, ul/ml

FIG. 4. (A) Polarity of subtilisin's active site as indicated by the hyperfine splitting constant AO ofthe active-site bound spin label. For a given spectrum, the best-fit simulation was independent of the starting values. Triplicate experiments produced a comparable change of AO values over the range of water added with a standard deviation of the initial AO value (at 0 AI/ml) of less than 1%. (B) AO values of the free spin label in tetrahydrofuran as a function of added
water.

molecules screen electrostatic interactions between polar and charged residues of the protein and hence induce greater freedom of motion. It is interesting to note that a similar "loosening up" of initially dry lysozyme, induced by partial hydration, was necessary for the enzyme to become catalytically active (4, 10). The subsequent leveling off of polarity at about 15% (wt/wt) bound water (found by inspection of Figs. 1 and 4) presumably corresponds to the formation and growth of water clusters (small patches of condensate) within the active site. At this point the transesterification activity has begun to decline. At water concentrations above 15% wt/wt and up to monolayer coverage [estimated to be ca. 35% (wt/wt)], water binds to less strongly interacting sites on the protein (e.g., nonpolar atoms not adjacent to the earliest forming clusters). This additional hydration is accompanied by a further increase in enzyme flexibility and, apparently, by a minor conformational change that causes the decrease in transesterification activity. Thus, our results suggest that lyophilized subtilisin does not have the same conformation as the partially hydrated enzyme, and that the initial increase in transesterification activity resulting from increased flexibility is offset by a conformational change at higher (albeit submonolayer) hydration levels. In this respect, subtilisin appears to behave differently from lysozyme, which has been reported to maintain its conformation during hydration (5). However, evidence that lysozyme does experience a conformational

change upon hydration has been presented by Baker et al. (8). In summary, enzymes suspended in organic solvents represent a versatile system for studying water-protein interactions and the involvement of water in enzyme structure and function. In particular, our results indicate a correlation between protein flexibility and catalytic activity; however, loss of certain activity can occur as the enzyme becomes increasingly flexible in a nonaqueous environment. In the case of subtilisin, it appears that the conformation of the lyophilized enzyme changes upon hydration, and that the conformational change accompanies the condensation of water over the protein.
We thank J. P. Klinman and R. J. Linhardt for helpful comments. This work was supported by the National Science Foundation (Presidential Young Investigator Awards to D.S.C. and J.S.D.), Genencor International, Mead Corporation, Merck and Co., and Olin Corp. R.A. was supported by National Institutes of Health Training Grant GMO8352. V.S. was supported by a Merck fellowship. 1. Tanford, C. (1%1) Physical Chemistry of Macromolecules (Wiley, New York). 2. Schultz, G. E. & Schirmer, R. H. (1979) Principles of Protein Structure (Springer, New York). 3. Bone, S. & Pethig, R. (1982) J. Mol. Biol. 157, 571-575. 4. Finney, J. L. & Poole, P. L. (1984) Comments Mol. Cell. Biophys. 2, 129-151. 5. Rupley, J. A., Gratton, E. & Careri, G. (1983) Trends Biochem. Sci. 8, 18-22. 6. Careri, G., Gratton, E., Yang, P.-H. & Rupley, J. A. (1980) Nature (London) 284, 572-573. 7. Careri, G., Giansanti, A. & Rupley, J. A. (1986) Proc. NatI. Acad. Sci. USA 83, 6810-6814. 8. Baker, L. J., Hansen, A. M. F., Rao, P. B. & Bryan, W. P. (1983) Biopolymers 22, 1637-1640. 9. Schinkel, J. E., Downer, N. W. & Rupley, J. A. (1985) Biochemistry 24, 352-366. 10. Rupley, J. A., Yang, P.-H. & Tollin, G. (1980) ACS Symp. Ser. 127, 111-132. 11. Careri, G., Giansanti, A. & Gratton, E. (1979) Biopolymers 18, 1187-1203. 12. Bone, 5. (1987) Biochim. Biophys. Acta 916, 128-134. 13. Dordick, J. S. (1989) Enzyme Microbiol. Technol. 11, 194-211.
14. 15.

Shown in Fig. 4A are AO values of the fluorophosphonate spin label linked to the active-site seine of subtilisin. These values were determined from spectra recorded at 120 K in tetrahydrofuran containing added water from 0 to 70 t.l/ml. Therefore, the AO values reflect the relative polarity of the active-site environment under these conditions. A larger AO corresponds to a higher polarity, and the increase from about 15.3 to 15.7 G is significant. For example, Fig. 4B shows the change of AO as water is added to tetrahydrofuran containing the free spin label. During AO measurements of the free spin label in various solvents, AO increased by 0.3 G when the solvent was changed from benzene to dimethyl sulfoxide (a change in dielectric constant from 2.3 to 47). Thus, Fig. 4 illustrates that the local polarity increases sharply as water is added up to 5 Al/ml of solvent, possibly because of water molecules binding in or near the active site. As more water is added to the solvent, a more gradual increase in AO is observed up to 20 dul of added water per ml of solvent, and above this water content, little increase in active-site polarity is evident. Interestingly, the initial increase in polarity coincides with the increase in transesterification activity (Fig. 2). Together, the activity results and the ESR data suggest the following sequence of events, which is similar to the stepwise process of protein hydration originally proposed by Rupley et al. (10). Initially, in the absence of added water, the lyophilized protein is very rigid and has relatively low catalytic activity. The adsorbed water [ca. 9o (wt/wt)] is strongly bound to ionizable groups on the protein. As water is added to the organic solvent, the active-site polarity increases because of further hydration of polar and charged groups in the active site. During this process, the active-site flexibility increases, as does the transesterification activity. The increased flexibility with increasing water content is consistent with Bone's hypothesis (3, 12) that protein-bound water

Zaks, A. & Russell, A. J. (1988) J. Biotechnol. 8, 259-270. Klibanov, A. M. (1990) Acc. Chem. Res. 23, 114-120.

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

20. Morrisett, J. D. & Broomfield, C. A. (1972) J. Biol. Chem. 247, 7224-7231. 21. Thomas, D. D., Dalton, L. R. & Hyde, J. S. (1976) J. Chem. Phys. 65, 3006-3024. 22. Atherton, N. M. (1973) Electron Spin Resonance (Wiley, New York). 23. Freed, J. H. (1976) in Spin Labeling: Theory and Applications, ed. Berliner, L. J. (Academic, New York), pp. 53-132.

16. Zaks, A. & Klibanov, A. M. (1988) J. Biol. Chem. 263, 31943201. 17. Ryu, K. & Dordick, J. S. (1989) J. Am. Chem. Soc. 111, 8026-8027. 18. Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B. & Power, S. (1988) J. Biol. Chem. 263, 7895-7906. 19. Himmelbrau, D. M. (1970) Process Analysis by Statistical Methods (Wiley, New York).