Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 Å resolution

R.K. Singh1, S. Gourinath1, S. Sharma1, I. Roy2, M.N. Gupta2, Ch. Betzel3, A. Srinivasan1 and T.P. Singh1,4

1 Department of Biophysics, All India Institute of Medical Sciences,New Delhi-110029, 2 Department of Chemistry, Indian Institute of Technology, New Delhi-11016 and 3 Institute of Medical Biochemistry and Molecular Biology, UKE, C/O DESY, Notkestrasse 85, 22603 Hamburg, Germany


    Abstract
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 Abstract
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 Materials and methods
 Results and discussion
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Three-phase partitioning is fast developing as a novel bioseparation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. Despite all this, the enzyme behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and the mixture was incubated at 25°C for 1 h. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The specific activity of the processed enzyme was estimated and was found to be 210% of the original enzyme activity. In order to understand the basis of this remarkable enhancement of the enzyme activity, the structure of the TPP-treated enzyme was determined by X-ray diffraction at 1.5 Å resolution. The overall structure of the TPP-treated enzyme is similar to the original structure in an aqueous environment. The hydrogen bonding system of the catalytic triad is intact. However, the water structure in the substrate binding site has undergone a rearrangement as some of the water molecules are either displaced or completely absent. Two acetate ions were identified in the structure. One is located in the active site and seems to mimic the role of water in the enzyme activity and stability. The other is located at the surface of the molecule and is involved in stabilizing the local structure of the enzyme. The most striking observation in respect of the present structure pertains to a relatively higher overall temperature factor (B = 19.7 Å2) than the value of 9.3 Å2 in the original enzyme. As a result of a higher B-factor, a number of residues, particularly their side chains, were found to adopt more than one conformation. It appears that the protein exists in an excited state which might be helping the enzyme to function more rapidly than the original enzyme in aqueous media. Summarily, the basis of increased enzymatic activity could be attributed to (i) the presence of an acetate ion at the active site and (ii) its excited state as reflected by an overall higher B-factor.

Keywords: acetate ion/crystal structure/enzyme activity/proteinase K/three-phase partitioning


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The analysis of enzyme behaviour in anhydrous media has important implications in biotechnology and bio-organic chemistry (Gupta, 1992Go; Halling, 1997Go; Tuena de Gomez-Puyou and Gomez-Puyou, 1998Go; Travis, 1993Go). At present, the interactions between protein and organic solvent are inadequately understood (Gregory, 1995Go; Gupta et al., 1997Go). A clear understanding will facilitate the use of a non-aqueous milieu for many applications such as organic synthesis, biosensors, bioseparation (Koskinen and Klibanov, 1996Go; Gupta, 2000Go) and perhaps enhancement of the enzyme activity and stability. Three-phase partitioning (TPP), a novel bioseparation strategy, is one such process. A scalable method, it is in fact useful as both an upstream and a downstream method in the production of enzyme proteins. Kosmotropy, electrostatic forces, conformation tightening and protein hydration shifts have been suggested as the physico-chemical basis for the protein coming out as an insoluble phase (Lovrein et al., 1987Go; Dennison and Lovrein, 1997Go). One of the main assumptions has been that the tert-butanol (the solvent which has been used most frequently) binds to the protein interior (Lovrein et al., 1987Go; Dennison and Lovrein, 1997Go). Our results with proteinase K (PK) show that this binding is not the cause of either enzyme being forced out of the aqueous phase or the remarkably enhanced biological activity observed after the enzyme has been subjected to TPP. We report here the results on the enzyme, which has been subjected to TPP, which clearly indicate the remarkable increase in the catalytic activity of PK. These results are supported by changes that occurred in the enzyme structure due to its treatment by TPP.


    Materials and methods
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 Materials and methods
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 References
 
Casein was a product of Sisco Research Laboratories (Mumbai, India). PK was obtained from Merck (Darmstadt, Germany). Bovine serum albumin was purchased from Sigma Chemical (St. Louis, MO). tert-Butanol was of HPLC grade and all other chemicals were of analytical grade and were procured locally.

Enzyme assay and protein determination

The commercially obtained PK was saturated with calcium chloride. It was purified by gel filtration on a Sephadex G-75 column. Fractions of highest activity were pooled, dialysed exhaustively against 0.05 M acetate buffer at 4°C and lyophilized. The caseinolytic activity of PK was estimated by following the increase in the amount of TCA-soluble peptides (Fujimura et al., 1987Go). The amount of TCA-soluble peptides was measured by Lowry's method (Lowry et al., 1951Go). The amount of protein was determined by the biuret method (Darbre, 1986Go) using bovine serum albumin as a standard protein. The purified enzyme showed a clean single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE).

Active site titration

The active site concentration of PK was determined using p-nitrophenyl acetate (pNPA) as a titrant. The enzyme concentration was calculated using an absorption coefficient of A1%280 nm = 14.2 (Ebeling et al., 1974Go). The reaction was carried out in 0.1 M sodium barbiturate buffer, pH 7.8, in a double-beam spectrophotometer. Freshly prepared pNPA (in acetonitrile) was added to both cuvettes and the increase in absorbance at 410 nm was measured. Using a value {varepsilon}410 nm = 16 595 for pNP, the active site concentration of the PK was calculated. The active site concentrations showed that the untreated enzyme preparation was 89.4% pure, whereas the samples of the treated enzyme corresponded to 91.2% purity.

Three-phase partitioning of proteinase K

A 3 ml volume of PK solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and incubated at 25°C for 1 h. The solution was then centrifuged (1800 g, 5 min). The lower aqueous layer and the upper organic layer were removed. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The dissolved PK was again saturated with calcium chloride and was purified by gel filtration on a Sephadex G-75 column. Fractions of highest activity were pooled, dialysed exhaustively against 0.05 M acetate buffer at 4°C and lyophilized. The purified samples of treated PK indicated a clear single band on SDS–PAGE. The experiment was also repeated with cacodylate buffer under similar conditions. The enzyme activity and the protein content were determined. Each experiment was carried out six times. The differences in the individual readings in each set were <3%. These data are listed in Table IGo.


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Table I. Three-phase partitioning (TPP) of proteinase K
 
Crystal preparation

PK (EC 3.4.21.14) from the fungus Tritirachium album Limber (Ebeling et al., 1974Go), obtained from Merck, treated by TPP and purified as described above,was crystallized by dissolving 10 mg of the lyophilized enzyme in 1 ml of 50 mM Tris–HCl, 1 mM CaCl2, pH 6.5. Drops of 25 µl of this solution were equilibrated in microdialysis setups against 1 M NaNO3 in the same buffer at 4°C. Single crystals of size 0.5x0.4x0.3 mm grew within 6–7 days. For checking the activity of the crystalline PK, a number of crystals were taken from the crystallization dish, crushed and dissolved in the same buffer to determine the activity in a similar manner as was done for the untreated and treated samples. The measurements were repeated six times. These values are given in Table IGo. This sample also corresponded to a single band on SDS–PAGE. In a further experiment to obtain a standard structure of PK under identical conditions for structural comparisions, the samples of the purified untreated enzyme were also crystallized in a similar manner.

X-ray diffraction data

For X-ray intensity data collection, one crystal of the treated samples was mounted in a glass capillary. The X-ray intensities were collected to 1.5 Å resolution at 12°C using a 300 mm MAR Research imaging plate scanner mounted on a Rigaku RU-200 rotating-anode X-ray generator operating at a rate of 40 kV and 100 mA. A graphite monochromator was used to produce Cu K{alpha} radiation. Crystallographic data, data collection and processing details are given in Table IIGo. The data were processed using the HKL package (Otwinowski and Minor, 1993Go). The B-factor was estimated from a Wilson plot (Wilson, 1942Go) to be 22.1 Å2. Two more data sets were collected on two further crystals of treated PK at different times. The overall B-factors estimated from the Wilson plot were 21.5 and 20.4 Å2, respectively. Another set of intensity data was also obtained on the freshly grown crystals of untreated PK under similar conditions. The overall B-factor calculated from the Wilson plot for this data set was 9.8 Å2.


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Table II. Overall statistics of data processing
 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Structure determination

The initial phases were determined by molecular replacement using the program AMORE (Navaza, 1994Go) as implemented in the CCP4 package (Collaborative Computational Project No. 4, 1994). The previously determined structure of PK at 1.5 Å resolution in aqueous buffer was used as the search model (Betzel et al., 1988Go). The rotation search using the 10.0–4.0 Å data revealed one unique solution. Comparison of the molecular replacement solution with that of the original search model showed that the root mean square (r.m.s.) displacement was only 0.56 Å, which indicated that no large structural rearrangement took place as a result of treatment of the enzyme by TPP. A series of 2FoFc maps were calculated using the phases determined from models that had sequential regions omitted (8% of the total atoms). The omit maps showed clear density for all the omitted regions.

Refinement and model building

For the refinement of this structure, PROTIN/PROLSQ programs of the CCP4 package (Collaborative Computational Project No. 4, 1994) were used so that the comparisions with the existing PK structures, which were earlier refined using these packages, could be based on similar calculations. After molecular replacement, the R-factor for the search model was 0.311 in the range 17.0–1.5 Å resolution and Rfree, consisting of reflections that were left out of the refinement (2% of the total), was 0.341. The search model was first rebuilt residue by residue into the omitted regions by using the omit maps generated as above, adjusting side chain torsion angles and occasionally residue rotamers. Approximately 40% of the residues required some adjustments to centre them in the electron density. The side chains of three amino acid residues, Gln103, Arg167 and Gln278, were not present in the original structure but were present in the structure of the untreated enzyme which was refined based on recent data. These side chains were also clearly visible in the structure of the treated enzyme and were fitted into the electron density well. The most striking observation was made in respect of a number of residues for which electron densities suggested multiple conformations of the side chains by rotations along the C{alpha}–Cß bond (Figure 1Go). These observations were confirmed by calculating omit maps. Several rounds of refinement by restrained parameter least squares using the PROTIN/PROLSQ program followed by manual rebuilding using the program O (Jones et al., 1991Go) were then performed on the protein structure. Initially, the model structure was used for 50 cycles of xyz refinement. Thereafter, the refinement was carried out for xyz and individual B-factors for another 30 cycles. During the refinement, adjustments of the side chains were made. The inspections of 2FoFc and Fo Fc difference Fourier maps indicated the positions of two Ca2+ ions, which were added in the subsequent cycles of refinement with occupancies of 1.0 and 0.5, respectively. The final B-values for these atoms were 11.1 and 15.1 Å2, respectively. The occupancy values for calcium in the other two structures of treated proteinase K were 1.0, 0.4 and 1.0, 0.5 with corresponding B-factors of 12.1, 13.4 and 12.4, 12.8 Å2, respectively. In the case of untreated enzyme, the values for the occupancy and B-factors were 1.0, 0.4 and 11.0, 12.1 Å2, respectively.




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Fig. 1. The residues showing two conformations for their side chains: (a) Thr4, (b) Ile13, (c) Ile107, (d) Ser130, (e) Ser140, (f) Ser143, (g) Ser219 and (h) Val230.

 
Solvent molecules

Two further rounds of manual building using O and refinement with PROTIN/PROLSQ resulted in the addition of 100 water molecules. Potential water molecules were located with the FoFc electron density map at the 3.0{sigma} contour level. Initially, they were only built into the density peaks that clearly exhibited the appropriate shape; any peaks that were possibly due to unknown molecules were excluded. Those water molecules were retained for which the electron density in the subsequent 2Fo Fc maps persisted after the refinement cycles and if they fulfilled the following criteria: within 3.4 Å of the enzyme oxygen or nitrogen atom (or bound water in the previous round) with good hydrogen bonding geometry, B-factors <45 Å2 and the real space correlation coefficients >65%. After inclusion of these 100 water molecules, the R-factor and Rfree came down to 0.233 and 0.264, respectively, for all the data (17.0–1.5 Å resolution). Further rounds of manual building in O combined with several cycles of refinement with PROTIN/PROLSQ resulted in the inclusion of 80 additional water molecules to a total of 180 water molecules in the model. The refinement with further cycles and model building resulted in the inclusion of a final 64 water molecules with an improvement in the final R-factor and Rfree to 0.193 and 0.206, respectively.

In the course of the refinement, two acetate molecules were identified. Acetate molecules were distinguished from water molecules in the electron density map because at the 1.0{sigma} contour level, there was electron density which was clearly triangular in appearance (Figure 2Go), whereas for water molecules electron densities were spherical. The existence of PK-bound acetate ions was further confirmed when we overlaid the 2Fo Fc electron density map for the enzyme in water with the refined coordinates of the TPP-treated PK and found no electron density corresponding to acetate ions.



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Fig. 2. The FoFc map at the 2{sigma} level showing the shapes of electron density for (a) water molecules and (b) acetate ions.

 
The refinement was also pursued for the data set from the untreated PK. The refinement converged well and the average B-factor for all the atoms was found to be 9.5 Å2. There were no clear split densities for the amino acid side chains except Asp207.

Model quality

The final model (Figure 3Go) containing 2030 protein atoms, two calcium ions, 244 water molecules and two acetate ions (eight atoms) gave an R-factor of 0.193 for all the reflections to 1.5 Å resolution. Rfree was 0.206 for 2% (688) of reflections. The model has a good stereochemistry with r.m.s deviations of 0.005 Å for bond lengths, 1.3° for bond angles and 24.7° for dihedral angles. The main- and side-chain atoms have well defined electron densities (Figure 4Go). Some 88% of the residues were located in the most favoured regions and 12% were found in the additionally allowed regions of the Ramachandran plot (Ramachandran and Sasisekaran, 1968Go) as calculated using the program PROCHECK (Laskowski et al., 1993Go). The quality of the model is summarized in Table IIIGo.



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Fig. 3. Three-dimensional folding of treated proteinase K. The active site residues Asp39, His69, Ser224 and acetate ions (Ac1 and Ac2) are indicated by ball and stick models. The figure was drawn using MOLSCRIPT (Kraulis, 1991Go).

 


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Fig. 4. Final 2FoFc map showing a section of electron density drawn at contour level of 1.0{sigma}. The electron density for the only water molecule in this region is also shown.

 

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Table III. Summary of crystallographic refinement
 
Binding of acetate molecules to the enzyme

There are two acetate molecules in the structure. The water molecule which is known to occupy the position of the oxyanion hole in the untreated PK (Betzel et al., 1988Go) structure and interacts with Asn161, Ser224 is absent from the present structure (Figure 5Go). Yet another water molecule in the untreated structure, which interacts with the previous water molecule and His69, is also absent in the structure of treated PK. In fact, the position of the latter was very close to the acetate ion in the present structure (Figure 5Go). It is noteworthy that the acetate ion in the present structure interacts with both His69 and Ser224 simultaneously and perhaps does the same job as is done by two water molecules in the aqueous medium. In the case of trypsin, a sulphate ion (Huber and Bode, 1978Go) and an acetate ion (Johnson et al., 1999Go) were observed. In the present structure the second acetate molecule was observed at the surface of the enzyme (Figure 6Go) and contributes to the stability of the molecule locally.



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Fig. 5. The interactions of (a) water molecules in PK (Betzel et al., 1988Go) and (b) water molecule and acetate ion in PKP (present structure). The dotted lines indicate hydrogen bonds. The distance between acetate C of CH3 and Met225 C{delta} (C{delta}H3) is 3.6 Å.

 


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Fig. 6. The acetate ion on the surface of the molecule showing perfect electron density 2FoFc at the 1.5 {sigma} level. The acetate ion is loosely bound and interacts mainly with solvent water molecules.

 
Structural comparison

The present structure (PKP), untreated PK in aqueous media (PKN) (Betzel et al., 1988Go), the currently determined structure of untreated PK in aqueous media under identical conditions (PKS) (T.P.Singh, 2000, unpublished results) and heat-treated PK in anhydrous media (PKT) (Gupta et al., 2000Go) were found to be similar with r.m.s shifts for the backbone atoms under 0.5 Å. Since the activity of PKP has increased 2.1-fold, attention was focused on the binding region of the enzyme. The substrate recognition site is formed by two peptide chains, Asn99–Ile108 and Leu131–Gly136. The residues of the recognition site extend from the catalytic site (Asp39, His69 and Ser224) to the surface of the molecule. The residues of the catalytic triad were found to have identical geometry in PKN, PKS, PKT and PKP within r.m.s. shifts of 0.4 Å, indicating no change in the stereochemistry of the catalytic site. The most striking change in the PKP was found in the conformations of the side chains of a number of residues, Thr4, Ile13, Ile107, Ser130, Ser140, Ser143, Ser219 and Val230 (Figure 1Go). All these residues had more than one conformational state for their side chains. It is noteworthy that several of these residues belong to the recognition site. On the other hand, in PKT, the conformation of only Ile107 changed with a rotation of 180° about the C{alpha}–Cß bond whereas in PKS, only Asp207 indicated multiple conformations, and in PKN no residue was found with more than one conformation. The conformation in PKT was stabilized through the interaction with a trapped acetonitrile molecule in the recognition site.

The overall temperature factor of 19.7 Å2 in the PKP is nearly twice as large as those observed in the untreated PKN (9.3 Å2) and PKS (9.5 Å2). In fact, this is the highest value of the B-factor observed so far for any PK stuctures (Table IVGo). Although the value of the B-factor may be influenced by a number of factors such as data processing and refinement protocols, a high-resolution structure with careful calculations reduces these effects to the minimum. Also, the observation of multiple conformations of the side chains of a number of residues in the present structure is indicative of the existence of a more flexible structure. Thus, to an extent, the increase in the value of the B-factor seems to have been caused by the treatment of PK under triphasic conditions. It can be stated further that the increased flexibility of the molecule as reflected in the form of B-factors is presumably responsible for the higher activity of the enzyme. Therefore, the treatment, which can stabilize the structure of an enzyme at higher temperatures, may enhance the enzyme activity considerably. Hence TPP appears to be a useful method to improve the purity and efficiency of enzymes. This is of great commercial significance and needs to be exploited further.


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Table IV. Average B-factors in various structures of proteinase K
 
Coordinates

The coordinates have been deposited in the Protein Data Bank with code number 1EGQ.


    Notes
 
4 To whom correspondence should be addressed. E-mail: tps{at}medinst.ernet.in Back


    Acknowledgments
 
Financial assistance from the Council of Scientific and Industrial Research (New Delhi) is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received May 4, 2000; revised February 1, 2001; accepted February 15, 2001.