Investigation of lipase-catalysed hydrolysis of naproxen methyl ester: use of NMR spectroscopy methods to study substrate-enzyme interaction.

Article abstract of DOI:10.1016/S0045-2068(02)00014-7

(+/-)-2-(6-Methoxy-2-naphthyl)propionic acid methyl ester (methyl ester of Naproxen), the precursor of therapeutically important nonsteroidal anti-inflammatory drugs (NSAIDs) was enantioselectively hydrolysed using as biocatalyst Candida rugosa lipase. In research aimed at studying the structure-activity relationship (SAR), NMR spectroscopy methods were employed to identify which Naproxen molecular moiety was essential to the substrate-enzyme interaction. The experimental results, in agreement with previous computer modelling studies and reported kinetic data, gave new information on the enzyme-substrate complex formation in solution.

Full text of DOI:10.1016/S0045-2068(02)00014-7

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 30 (2002) 276–284
www.academicpress.com
Investigation of lipase-catalysed hydrolysis of
naproxen methyl ester: use of NMR
spectroscopy methods to study
substrate–enzyme interaction
E. Cernia, M. Delfini, E. Di Cocco, C. Palocci, and S. Soro*
Department of Chemistry, University of Rome ‘‘La Sapienza,’’ Piazzale Aldo Moro 5,
Rome 00185, Italy
Received 28 November 2001
Abstract
(
ꢀ)-2-(6-Methoxy-2-naphthyl)propionic acid methyl ester (methyl ester of Nap-
roxen), the precursor of therapeutically important nonsteroidal anti-inflammatory
drugs (NSAIDs) was enantioselectively hydrolysed using as biocatalyst Candida
rugosa lipase. In research aimed at studing the structure–activity relationship (SAR),
NMR spectroscopy methods were employed to identify which Naproxen molecular
moiety was essential to the substrate–enzyme interaction. The experimental results, in
agreement with previous computer modelling studies and reported kinetic data, gave
new information on the enzyme–substrate complex formation in solution. Ó 2002
Elsevier Science (USA). All rights reserved.
Keywords: Hydrolysis; Lipase; Naproxen; Structure–activity relationship (SAR); NMR
relaxation times
*
Corresponding author. Fax: +39-06-49913340.
E-mail address: simonetta.soro@uniromal.it (S. Soro).
0
045-2068/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S0045-2068(02)00014-7

E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
277
1
. Introduction
Hydrolases and in particular microbial lipases have found widespread appli-
cation as biocatalysts for the solution of synthetic organic problems, in particular
in the field of regio- and enantioselective reactions [1,2]. Due to their role in
nature as catalysts for the hydrolysis of triglycerides (fats and oils), lipases are
ubiquitous in the world of mammals, plants, and micro-organisms such as bac-
teria and fungi. Although lipases have been designed by nature for the hydrolysis
of triglycerides, they are also capable of catalysing the selective hydrolysis and
synthesis of unnatural esters with widely differing structures without requiring
cofactors. This is also the reason for their widespread application in organic
synthesis.
In the last decade the crystallographic data for several enzymes have became
available [3–5] providing three-dimensional structures of their active sites. These
findings, together with the development of molecular modelling software capable
of investigating on the interactions between the lipase amino acid catalytic site and
different substrates, offered the possibility of rationalising the lipase–substrate in-
teraction on the basis of the experimental results obtained. On the other hand, in
1
solution H-NMR relaxation rate measurements of substrate–lipase complexes
yield the evaluation of the motional and environmental features of bonded sub-
strate. Selective irradiation methods can be used throughout the experiments since
the selective proton relaxation rate (R
correlation time and hence by enzyme-binding interactions, as shown elsewhere
6,7].
Candida rugosa lipase (CRL), one of the most synthetically useful lipase, was
s
) is linearly affected by changes in motional
[
employed as catalyst in the enantioselective hydrolysis of (ꢀ)-2-(6-methoxy-2-
naphthyl)propionic acid methyl esters (methyl ester of Naproxen).
This compound, which belongs to the class of nonsteroidal anti-inflammatory
drugs (NSAIDs) is used in the treatment of rheumatoid arthritis and osteoarthritis
[
8]. Only one enantiomer of this class of drugs usually show biocatalytical activity, in
particular the S enantiomer of Naproxen is 28 times more active than the R isomer
as an anti-inflammatory agent [9].
Here we present NMR data related to the interaction of (ꢀ)-2-(6-methoxy-2-
naphthyl)propionic acid with CRL supporting the kinetics hydrolysis experiments.

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E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
2
. Materials and methods
2
.1. Enzyme and substrates
Methyl ester of (ꢀ)-2-(6-methoxy-2-naphthyl)propionic acid was obtained from
Alfa Wassermann (Karlsrhue, Germany); tributyrin and lipase from C. rugosa (Type
VII) (commercial-CRL) were purchased from Sigma (St. Louis, MO, USA); another
lipase from C. rugosa obtained by fermentation in special conditions was the gift of
Prof. F. Valero, ‘‘Universitat Autonoma de Barcelona’’ (UAB-CRL).
2
.2. Enzymatic reactions
The lipolytic activity was assayed by alkalimetric final titration. The assay mix-
ture, containing 2.5 ml buffer (20 mM phosphate buffer, pH 7.5), 0.5 ml tributyrin,
and 0.1 ml enzymatic solution (150 mg/ml), was incubated at 37 °Cunder magnetic
stirring (300 rpm) for 30 min. The reaction mixture was stopped with 2.5 ml acetone/
ethanol mixture 1:1 (v/v) and titrated with 0.05 M NaOH in the presence of
phenolphthalein as indicator using an automatic burette (Methrom). The protein
content was measured with the Sigma kit (Lowry method).
Methyl ester was hydrolysed employing essentially the same procedure reported
by Sih et al. [10]. To 100 mg crude CRL in 1 ml of 0.2 M phosphate buffer, pH 7.9,
0
.5 mmol ester was added. The resulting suspension was stirred vigorously at 40 °C
for 168 h and then centrifuged for 20 min at 600 rpm. The precipitate was washed
with 0.2 M phosphate buffer and centrifuged again to collect the water insoluble
ester. Supernatant and washing were combined and acidified to pH 2.0 with HCl and
the precipitate was collected by filtration to yield the acid.
No hydrolysis was observed at pH 7.5 and 40 °Cin the absence of the enzyme.
2
.3. Analytical methods
Enzymatic conversion of methyl ester of (ꢀ)-2-(6-methoxy-2-naphthyl)propionic
acid to the corresponding acid and enantiomeric excesses (e.e. %) were determined
by enantioselective normal-phase high-performance liquid chromatography (NP-
HPLC). Analytical liquid chromatography was performed on a Waters chro-
matograph equipped with a Rheody Model 7725i 20 ll injector and two Model
M510 solvent-delivery systems. The detector used was a Model M490 program-
mable multi-wavelength detector (Water Chromatography, Division of Millipore,
Milford, MA). Chromatographic data were collected and processed using the
Millennium 2010 Chromatography Manager software (Waters Chromatography).
The column was a commercially available Chiralcel OD-H chiral stationary phase,
2
2
5cm ꢁ 4:6 mm i.d. (Chemical Industries, Tokyo, Japan). The mobile phase was
% 2-propanol in n-hexane (v/v), at a flow rate of 1.0 ml/min. Retention times
obtained for the considered compounds were the following: T
of Naproxen, 4.87 min; T
ðRÞ-methyl ester of Naproxen, 5.21 min. Chromato-
graphic runs were all monitored by UV detector at 254 nm, at temperature of
5 ꢀ 1°C.
1
ðSÞ-methyl ester
2
2

E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
.4. NMR studies
Phosphate buffer solutions were prepared in deuterium oxide (Sigma, St. Louis,
279
2
MO, USA) at pH 7.4, and carefully deoxygenated by three freezing vacuum pumping
thawing cycles, immediately followed by sealing off the NMR tube.
All NMR experiments were carried out on a Bruker AM-500 and on a Varian XL
3
referenced to external [ H ]trimethylsilylpropanesulfonate.
00 spectrometers at the controlled temperature of 300 ꢀ 1 K. Chemical shifts were
2
4
Experimental conditions on Bruker AM-500 were: 90° pulse length, 9.0 ls; 12
different s values from 1 ms to 40 s; repetition rate, 40 s. Experimental condition on
Varian XL 300 were: 90° pulse length, 8.5 ls; 12 different s values from 1 ms to 35 s;
repetition rate, 35 s.
Proton spin–lattice relaxation rates were measured with inversion-recovery pulse
sequences and calculated by exponential regression analysis of recovery curves of
longitudinal magnetisation components. Single and double selective proton spin–
lattice relaxation rates were measured with inversion-recovery pulse sequences im-
plemented with DANTE or double DANTE sequences. All relaxation rates were
calculated with initial rate approximation [11].
Assignments were performed by two-dimensional COSY experiments and are in
line with previous literature data [12].
3
. Results and discussion
3
.1. Kinetic data
Commercial and UAB lipases were characterised for their lipolytic activity and
protein content (Table 1). UAB-CRL showed higher activity and protein content
then commercial lipase. This finding was expected given the higher purity of UAB-
CRL preparation also in terms of isoform composition [13,14].
Kinetic experiments on (ꢀ)-2-(6-methoxy-2-naphthyl)propionate methyl ester in
hydrolysis reaction using both lipases as catalysts are reported in Fig. 1.
Table 1
Lipases activity
Lipase
Protein content (lg/ml)
Lipolytic activity (leq acid/min)
Commercial-CRL
UAB-CRL
18 ꢀ 5
3:7 ꢀ 0:1
100 ꢀ 3
11:4 ꢀ 0:1

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E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
Fig. 1. Hydrolysis reaction of Naproxen methyl ester (0.5 mM) catalysed by two preparation
of CRL (100 mg/ml) in buffer solution (pH 7.9). Conversion (%) versus reaction time.
The results showed UAB-CRL was more active than commercial-CRL both in
terms of conversion and enantiomeric excess. These data were in agreement with the
composition and lipolytic activity of the two different preparations. Moreover, the
low hydrolytic rate observed was typical of lipase-catalysed hydrolysis with synthetic
substrates and in dispersion systems [15].
3
.2. NMR data
The Naproxen proton chemical shifts, relevant assignments and proton relaxation
rates are reported in Table 2.
The relaxation values obtained were consistent with a relaxation mechanism
1
1
mainly determined by H– H dipolar interaction as confirmed by Rnsel=Rsel ratio.
Table 2
1
H NMR chemical shifts, nonselective relaxation rate (Rnsel), selective relaxation rate (Rsel),
relaxation rate fraction (F ¼ Rnsel=Rsel) for 1 mM Naproxen protons in deuteriumoxide–
phosphate buffer (0.1 M, pH 7.4, 300 ꢀ 1 K)
Ã
Ã
Ã
Ã
sel
d (ppm) Assignment
R
nsel
R
(free)
sel
F
(free)
R
nsel
R
F
(bound) (bound) (bound)
(
free)
7.80
7.77
7.71
7.44
7.31
7.17
3.89
3.74
1.44
8
0.66
0.65
0.86
0.58
0.68
0.38
0.74
0.54
1.58
1.48
1.43
0.49
0.44
0.67
0.65
0.87
nd
0.44
0.45
1.76
1.32
1.01
0.59
0.86
nd
0.62
0.63
0.62
0.56
0.68
0.42
0.76
0.60
1.56
1.63
2.72
0.76
0.98
1.14
0.66
1.14
nd
0.38
0.23
0.82
0.57
0.60
0.64
0.67
nd
4
1
3
5
7
OCH
CH
CH
3
3
1.74
0.91
2.08
0.75
*
Errors on the relaxation rates are less than 3%.

E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
281
The motional correlation times were evaluated by the dipolar interaction energy
between protons at fixed distances, rij as measured by double selective relaxation
rates, according to the equation
!
4
2
c ꢀh
ij ¼ R À R ¼ 0:1 r6
6sij
ij
1
sel
i
r
À sij
;
ð1Þ
x2s2
ij
ij
ij
1
where R is the double selective relaxation rate measured for H
i
upon selective ex-
sel
citation of H
the proton magnetogyric ratio, rij is the H
Larmor frequency, ꢀh is the reduced Planck’s constant and sij is the motional cor-
relation time characterising reorientation of the H –H vector.
i
and H
j
, Ri is the single selective relaxation rate measured for H
i
, c is
i
–H internuclear distance, x is the proton
j
i
j
In our study this correlation time cannot be identified with the molecular reori-
entational time because the aromatic moiety is very likely to undergo anisotropic
reorientation and the considered internuclear vectors may be rather affected by
internal motions.
Infact a certain contribution from a motional anisotropy can be devised since it is
known that aromatic molecules as 1,2-dichlorobenzene and 1,2,3-trichlorobenzene
exhibit different values for the motional correlation times along the cartesian axes [16].
Consequently, calculations on the basis of Eq. (1), and of the spin lattice relax-
ation times measurement at different fields [17] provided an effective correlation time
s values (Table 3). The s obtained for aromatic protons in absence of lipase showed
c
c
values in the range 0.55–0.75 ns. In presence of lipase (molar ratio Naproxen/lipase
00:1) the obtained s values were in the range 1.05–4.30 ns, with the higher increase
exhibited by H , H , and H protons. These data clearly indicated that the H , H
and H protons were the most involved in the enzyme interaction.
1
c
3
5
7
7
5
,
3
On addition of lipase up to lipase:ligand is 0.05 ratio, all chemical shifts were
almost unaffected whereas all proton relaxation rates were selectively enhanced, Rsel
being much more affected, as expected [6], than Rnsel
.
The selective relaxation rate enhancements could be studied at variable Naproxen
concentration while keeping the concentration of lipase constant. In principle such
effects might also be determined by the increased viscosity of the medium. However,
as already noted elsewhere [18] the fact that 1=DRsel shows linear dependence on the
ligand concentration at fixed value of protein concentration demonstrates that the
observed phenomena arise from binding to the protein, with consequent slowing
Table 3
Naproxen protons correlation times (s
c
) in absence (a) and in presence (p) of lipase
Ã
Ã
Proton number
s
c
(a) (s)
s
c
(p) (s)
À10
À10
À10
À10
À10
À10
À10
À10
À10
À10
À10
À10
8
4
1
3
5
7
6:0 ꢁ 10
6:0 ꢁ 10
5:5 ꢁ 10
7:0 ꢁ 10
7:0 ꢁ 10
7:5 ꢁ 10
13:5 ꢁ 10
13:5 ꢁ 10
10:5 ꢁ 10
23:0 ꢁ 10
18:5 ꢁ 10
43:0 ꢁ 10
*
À10
(s).
Error on s
c
values are less than 0:2 ꢁ 10

282
E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
5
Fig. 2. Behaviour of l=DRsel of Naproxen H proton as function of the substrate concentration
in presence of 1 mg/ml lipase in deuterium oxide phosphate buffer (0.1 M, pH 7.4, 300 ꢀ 1 K).
down of molecular motions. Moreover the occurrence of dipole–dipole interactions
with the enzyme was expected to contribute to the relaxation rate enhancement. The
5
variation of 1=DRsel for H proton is shown in Fig. 2 and allows the evaluation of the
apparent association constant (K ) by extrapolating data to 1=DRsel ¼ 0, where
ass
[
Naproxen] ¼ À1=K , according to the equation
ass
1
=DR ¼ ð1=K þ ½LꢂÞðC 1=R Þ;
i
ass
M
i;b
where Ri;b is the relaxation rate of H
i
bound, C
M
is the enzyme concentration, and
obs
i
free
DR ¼ R À Ri
.
i
3
À1
The calculated value, K ¼ 0:5 ꢁ 10 M , suggests that the Naproxen is not
ass
À1
tightly bound by lipase. The proton relaxation scale (ꢃ1 s ) and the observed
constant confirm fast chemical exchange of the ligand between the protein bound
environment and the bulk such that relaxation rates measured in the presence of
lipase are averaged according to the equation
sel
obs
sel
f
sel
b
R
¼ p R þ p R ;
f
b
ij
obs
ij
f
ij
b
r
¼ p r þ p r
f
b
the p fractions of ligand in each environment can be approximated by pb
bond) ¼ protein/ligand and p ðfreeÞ ¼ 1 À p ꢃ 1.
(
f
b
The selective relaxation rate enhancement depends on the increase in the motional
correlation time s as well as on the possible increased number of dipolar interaction
c
contributing to the relaxation rate. Such a rate was determined by reduced molecular
motions accompanied by dipole–dipole interactions with enzyme protons. It was
noticed that the aromatic ring protons were greatly affected, suggesting the mode of
binding.
The cross-relaxation rates measured in the presence of the enzyme provided a
means of improving the characterisation of the binding interaction, allowing the
calculation of the dipolar interaction energies of proton pairs in the bound state.

E. Cernia et al. / Bioorganic Chemistry 30 (2002) 276–284
283
The cross-relaxation rates change from relatively small positive to large negative
values and it is consistent with slowing down of molecular motion from a region
where xs
c
6 1 to one where xs ꢄ 1.
c
4
. Conclusions
Since its very first application NMR method has always attracted investigators to
the possibility of studying small molecules interacting with macromolecules; unfor-
tunately the NMR approach has always been limited by the exchange rate from the
bound state since only fast exchange yields sizeable changes in the NMR parameters.
Fast off-rates do not, of course, characterise the usual strong interactions with
substrates thus severely limiting NMR applications to systems of most importance.
Lipase, on the contrary, does allow NMR investigations of bound substrates, as
demonstrated by the present data.
The substrate exchange from the bound state at a rate fast enough to yield
measurable selective relaxation rate enhancements thus provides significant dynamic
and structural features.
In the case of the investigated substrate the interaction constant measured by
NMR relaxation data showed no tight binding between substrate and enzyme. This
is in agreement with reported kinetic data in hydrolysis reaction of Naproxen ester.
Moreover, the NMR relaxation data showed that only a portion of aromatic ring
protons was the most affected by lipase interaction. This kind of interaction between
substrate aromatic rings and enzyme was proposed in previous computer modelling
studies [19]. The SAR theoretical approach was able to rationalise substrate–enzyme
interactions based on enzyme three-dimensional crystal structure and docking
studies suggesting some amino acids (particularly Phe345) as playing an important
role in determining enzyme–substrate recognition. This finding was confirmed by
protein engineering studies [20].
In conclusion this study confirms the p interaction and highlights the substrate
moiety directly involved in the enzyme–substrate interaction in solution during the
catalytic process.
Acknowledgments
This work was financially supported by Programma Chimica and Programma
Biotechnologie (legge 95/95), CNR Italy. The authors are indebted to Dr. Fancisco
Valero for providing lipase and to Prof. Francesco Gasparrini for his help in
establishing the analytical methodologies.
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