1Centre for Biotechnology, Anna University, Guindy, Chennai 600 025, India, 2SMPE, Nanyang Technological University, Singapore 629798 and 3AU-KBC Research Centre, Anna University, Chrompet, Chennai 600 044, India
4 To whom correspondence should be addressed at: Centre for Biotechnology, Anna University, Tamil Nadu, Chennai 600 025, India. e-mail: pgautam{at}annauniv.edu, gpennathur{at}vsnl.net
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Abstract |
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Keywords: Candida rugosa lipase/enantioselectivity/ibuprofen esters/molecular dynamics simulations/non-steroidal anti-inflammatory drug
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Introduction |
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Lipases have also been useful as an industrial catalyst for the resolution of racemic acids and alcohols and in a variety of fields such as household detergents (Falch, 1991), oils and fats (Macrae, 1983
), dairy (Izco et al., 2000
), organic media (Cardenas et al., 2001
), leather and paper industries.
Ibuprofen is a non-steroidal anti-inflammatory drug and is generally administered as a racemate (Hutt and Caldwell, 1984). The inversion of the R() to S(+) that occurs for aryl-propionic acids may have both toxicological and therapeutic implications when the drug is administered as a racemate (Adam et al., 1976
; Beck et al., 1991
). Resolution of racemic ibuprofen by Candida rugosa lipase (CRL) can be achieved through stereospecific esterification in non-aqueous media (Mustranta, 1992
), while in aqueous media this approach is extended to stereospecific hydrolysis of the corresponding esters.
The factors that trigger the activation at the lipidwater interface have been discussed. It was suggested that a conformational change in the enzyme might lead to the increased activity (Sarda and Desnuelle, 1958). Using standard protein structure determination methods, it was found that there was a minimal structural change in lipases except at the flap region (comparison of open and closed forms with and without inhibitor state that explicitly). Maximum conformational change observed during activation involves a rigid body hinge type motion of a single helix (lid/flap) (Hermoso et al., 1997
).
The structure of CRL (Grochulski et al., 1993) revealed that the active site Ser209, His449 and Glu341 is covered by a polypeptide (lid) flap and is not exposed to the solvent. The activation of CRL requires the movement and the refolding, including a cis to trans isomerization of a proline residue of a single surface loop to expose a large hydrophobic surface to interact with the lipid interface (Grochulski et al., 1994
). The comparison of structures, of the open form and the closed form of lipase, revealed that the difference is in the movement of this flap (Cygler and Schrag, 1999
). The mechanism whereby CRL differentiates between the two antipodes of a chiral substrate was influenced by several physiochemical factors such as temperature (Holmberg and Hulk, 1991
), solvent hydrophobicity (Wu et al., 1990
; Lakshmi et al., 2000
) and hydrostatic pressure (Kamat et al., 1993
). The highly electronegative substituent (F, Cl) at the alpha carbon of the alcohol moiety increases the rate of CRL catalysed hydrolysis (Botta et al., 1996
).
A variety of triglycerol lipase structures have been solved (Brandy et al., 1990; Winkler et al., 1990
) and not all lipases have their active site shielded by a flap/lid, e.g. Bacillus subtilis (Pouderoyen et al., 2001
) lipase lacks the flap. Recently, the sequencing of Cephaloleia presignis (Espinosa et al., 2000
) lipase revealed that there was a very different initial sequence (first 28 amino acids from the N terminal) with respect to the many known lipases.
All the enzymes in water have a pH-activity profile. In porcine pancreatic lipase, it is important to have the enzyme in a buffer with the optimal pH for activity prior to dehydration for use in organic solvents with low or no buffering capacity (Zaks and Klibanov, 1985). The effect of pH in the water phase localized around the enzyme in organic solvents using hydrophobic esters of fluorescein as indicators (Brown et al., 1990
; Valivety et al., 1990
) has been studied. There have been very few reports concerning the effect of pH on the enantioselectivity of enzymes (Schnider et al., 1984
). Studies have concluded that pH can greatly influence the activity and the enantioselectivity of lipase, which might probably result from the conformational changes of lipase with pH variation. It was observed that the best enantioselectivity of CRL towards 2-chloroethyl ether of ketoprofen was obtained at pH 2.2 with the enzyme still active and stable (Liu et al., 1999
).The activity of human gastric lipase (HGL) with short chain and long chain triacylglycerols was determined as a function of pH (Gargouri et al., 1986
). The maximum specific activity was reached at pH 6.0 with tributyrin with long chain triacylglycerols and the maximum lipolytic activity was obtained in the pH range of 4.55.5. The influence of pH on the specific activities of rabbit gastric lipase (RGL) (Moreau et al., 1988
) and dog gastric lipase (DGL) (Carrière et al., 1991
) was also tested using short chain, medium chain and long chain triacylglycerols as substrates. The specific activity of DGL on long chain triacylglycerol (TAG) reached a maximum at pH 4.0, whereas it was 6.0 and 5.5 on short chain TAG with HGL and RGL. The medium chains were hydrolysed at an optimum pH of 6.0.
Molecular dynamics (MD) simulations have been used in analysing the conformations the solute acquired in various solvent conditions (Peters and Bywater, 1999).The residues in the flap play a major part in the activity of Thermomyces lanuginose lipase (Cajal et al., 2000
). Previous MD simulations studies performed on Rhizomucour miehei lipase (Peters et al., 1996a
,b; Peters and Bywater, 1999
) provided considerable insight into the lid dynamics and the possible manner in which the protein may accommodate incoming substrates. MD simulation studies have also been used to elucidate the effect of point mutation in Humicola lanuginosa (Peters et al., 1998
).
In this paper, we describe an interesting phenomenon that was observed as we were trying to enhance the yield of S(+) ibuprofen by the hydrolysis of its esters by CRL. We found that on varying the pH of the reaction mixture there was a differential selectivity of the enzyme for the substrate at two different pH conditions. We used MD simulations to study this differential substrate selection by CRL under varying conditions of pH and attributed it to the differential motion of the flap using MD simulations.
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Materials and methods |
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Hydrolysis reaction
Methyl ester of ibuprofen. Unless otherwise stated, the reaction mixture consisted of 10 mg of CRL dispersed in 10 ml of citric acid phosphate buffer (Mcilvaine buffer pH 5.6) and 20 ml of racemic ibuprofen ester. The mixture was agitated at 170 r.p.m. at 37°C for 5.5 h. A known aliquot of the reaction mixture was vortexed with twice the volume of n-hexane. Ibuprofen and ibuprofen ester, being immiscible in water and soluble in hexane, were extracted into the organic phase and 10 µl of the organic phase was analysed by HPLC.
1-Butyl ester of ibuprofen. Sodium phosphate buffer pH 7.2 was prepared and 50 mg of CRL was added to 10 ml of the buffer. The reaction was started by adding 1 mM of the substrate butyl ester of ibuprofen (262 mg). The mixture was kept at 37°C in a water bath shaker at 150 r.p.m. for 48 h.
Chiral analysis of S(+) ibuprofen
Methyl ester of ibuprofen. The extract was treated with sodium bicarbonate (0.5 M) solution to convert the acid (ibuprofen) to its sodium salt that later gets extracted in the aqueous phase, while the unreacted ester remained in the organic phase. The aqueous phase was treated with 3 N HCl to regenerate ibuprofen and the acidic solution was shaken with 1-hexane to extract the regenerated ibuprofen in the organic phase. The yield of S(+) ibuprofen was monitored by HPLC using a Chiral Diacel OD column capable of separating the R() and S(+) enantiomers. The mobile phase was hexane2-propanolTFA (100:1:0.1), the column temperature was 30°C and the flow rate was 1 ml/min.
1-Butyl ester of ibuprofen. The reaction mixture was extracted in two 5 ml portions of HPLC grade hexane. The organic layer was collected and to this 10 ml of 0.5 ml sodium bicarbonate (NaHCO3) was added and shaken well in a separating funnel. The aqueous layer was collected and acidified using 3 N HCl. The acidified NaHCO3 was re-extracted in 10 ml of hexane using a separating funnel. The aqueous layer was discarded and the organic fraction was dried over anhydrous magnesium sulphate (predried in an oven at 100°C overnight). The hexane was totally evaporated in a rotary evaporator; to this 1 ml of hexane was added, vortexed and used for the chiral analysis in HPLC. The enantiomer of ibuprofen was identified by analysing the samples in HPLC using a chiral column and comparing with the standard resolved enantiomer of ibuprofen. The mobile phase consisted of hexaneisopropanolTFA in the ratio of 100:1:0.1. The flow rate was set to 1 ml/min and the peaks were detected at 254 nm.
Molecular dynamics simulation
As a starting point for the simulations the coordinates of the open form of CRL (1CRL.pdb) was obtained from the Protein Data Bank (www.rcsb.org/pdb). The MD simulation was performed using GROMACS. The leap-frog algorithm for integrating Newton equations and the periodic boundary condition was applied. All bonds were constrained using LINCS (Hess et al., 1997) and 15 600 water molecules were added to 5126 and 5124 solute atoms and the box size was 8.77900x8.62200x7.27500 nm, at the end there were 51 929 and 51 927 atoms to be simulated in the acidic and neutral environments, respectively. During energy minimization the steepest descents algorithm was used and in both pH cases the minima was reached in 159 steps. MD was performed with a time step of 2 fs and the coordinates were saved every 1000 steps. The potential energy was conserved during the MD. The MD simulations were performed at 300 K and Berendsen temperature coupling (Berendsen et al., 1984
) was applied. MOLMOL (Koradi et al., 1996
) was used to calculate the root-mean-square deviation (r.m.s.d.) of the flap of CRL in the acidic and neutral conditions to the energy-minimized structure.
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Results |
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Huge-Jensen et al. (1987) reported that the hydrolysis of olive oil catalysed by commercial lipase is sensitive to the nature of buffer pH owing to an enzymebuffer interaction. The desired amount of active site protonation can be achieved by altering the pH of the reaction mixture. Hence, pH of the reaction mixture can play an important role in optimal enzyme activity. Generally, it is a well known fact that enzymes lose their activity under extreme pH conditions. Gu et al. (1986
) have shown that the enzymatic resolution of methyl ester of aryl propionic acids by CRL was slow at pH 8.0.
We have studied the enzymatic hydrolysis of methyl and butyl esters of ibuprofen at pH 5.6 and pH 7.2 using CRL. The two ester substrates differ in the nature of the alkyl group of the alcohol moiety. Interestingly, we found that CRL prefers the methyl ester of ibuprofen at pH 5.6 and the butyl ester of ibuprofen at pH 7.2 (Table I). The reactions proved to be highly enantioselective with only the S(+) ibuprofen esters getting hydrolysed. Table I shows the yield of the methyl and butyl esters of S(+) ibuprofen at different pH conditions taken as a measure of the rate of transformation. The observed (%) yield of S(+) ibuprofen in the CRL catalysed hydrolysis was found to decrease with increase in the chain length of the alkyl group at pH 5.6. The reverse is observed at pH 7.2 with preference being accorded to the butyl ester of S(+) ibuprofen.
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In order to understand the molecular basis of this phenomenon, we performed MD simulation of the open form of the CRL in different pH conditions using GROMACS force field. On analysis of the amino acid sequence of CRL, we identified all the titrable amino acid groups in the enzyme and compared their pKa values (Voet and Voet, 1995). Amino acids glutamic acid and aspartic acid have pKa values <5.0 and hence it is not right to protonate them for simulation at pH 5.6 nor at pH 7.2. There are five histidines in the amino acid sequence of CRL which can be protonated/deprotonated as a function of pH. The amino acid lysine has a pKa value of >10 and therefore all solvent-exposed lysines in the enzyme are heavily protonated at pH 7.2 and pH 5.6.
The pKa value of the lysines and other titrable residues was calculated using Delphi (Honig and Nicholls, 1995). It showed that the percentage protonation of lysines was 99 and 96% at pH 5.6 and 7.2, respectively. Hence, we proceeded to model CRL with the assumption that all the lysines are protonated immaterial of which pH state the enzyme is in (pH 5.6 or 7.2). To incorporate a pH gradient only the histidines were protonated at NE2 and ND1 sites to model pH 5.6 and 7.2.
After 2 ns of simulation at pH 5.6 and 7.2 the flap of CRL showed a r.m.s.d. of 3.048 Å, as shown in Figure 1a and b, to the energy-minimized conformer, but there was no differential movement of the flap in comparison with each other. This indicates that the differential pH-based substrate specificity of the enzyme towards butyl and methyl esters of ibuprofen cannot be explained in terms of protonation results given by the pKa predictors.
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Discussion |
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The two buried lysines (K180 and K404) are fully protonated at pH 5.6 as compared with pH 7.2, and thus exist with a perturbed pKa in their micro-environment. This was exploited in the modelling of the pH states. The buried lysines at positions 180 and 404 were protonated along with all the other lysines to simulate the behaviour of CRL at pH 5.6 (shown in Figure 2). These two residues were left un-protonated during simulation at pH 7.2 with all the other lysines protonated. The protonation states for histidines were assigned by the MD program (GROMACS) which computes the hydrogen-bonding pattern of the protein, and indicates if the proton should be on the NE2 rather than the ND1 site or at both ND1 and NE2 sites of histidine.
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Conclusion |
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From the simulation of CRL in a hydrophilic environment at two different pH states we see that the CRL flap moves towards the closed conformation in an acidic environment. This results in a decreased availability of the hydrophobic zone produced by the lesser extent of the opening up of the flap. We deduce that the conformation of the enzyme in an acidic environment is more favourable for smaller and less hydrophobic side chain substrates. In neutral environment, simulations show that the flap opens up more as compared with an acidic environment. The further opening of the flap produces a larger hydrophobic zone on CRL favourable for larger substrates with a more hydrophobic side chain to gain access to the active site. This leads us to extrapolate that the choice of substrate is very much dependent on pH for CRL. The motion of the flap is necessary for the substrate entering the active site.
Researchers worldwide are trying to understand the structurefunction relationship of lipases, especially CRL, and we hope to shed more light on the significant role played by pH. The theoretical studies done here by means of MD simulations have successfully elucidated why a more hydrophobic butyl ester of ibuprofen is preferred by CRL at a neutral pH and the methyl ester of ibuprofen at an acidic pH. This pH-based structurefunction relationship helps contribute to a better understanding of the functionality of CRL by probing the flap dynamics.
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Acknowledgements |
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Received March 27, 2003; revised October 27, 2003; accepted October 30, 2003