In the Laboratory
Regiospecific Ester Hydrolysis by Orange Peel Esterase An Undergraduate Experiment Timothy D.H. Bugg,* Andrew M. Lewin, and Eric R. Catlin Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK Enzyme catalysis is an important topic in undergraduate bioorganic chemistry courses, but it presents problems to demonstrate effectively in a chemistry laboratory. As an accompaniment to a second year undergraduate course in bioorganic chemistry, we have recently developed a simple but effective experiment that demonstrates the regiospecificity of enzyme catalysis using an esterase activity easily isolated from orange peel (1) and compares its specificity with that of two commercially available esterase enzymes. This experiment offers students the opportunity to see enzyme catalysis in action and to prepare an enzyme extract from a natural source. The experiment involves the synthesis and assay of diester derivatives of para-, meta-, and ortho-hydroxybenzoic acid (Fig. 1), in which the regiospecificity of enzymatic hydrolysis is demonstrated by the selective hydrolysis of one of the two ester functional groups. Typically a group of 4–6 students starts from the same hydroxybenzoic acid but each student uses different reagents, so that the group can synthesize a set of related diester derivatives. The enzymatic hydrolysis of each diester derivative is then assayed against orange peel esterase (as a crude extract) and commercially available pig liver esterase (PLE) and porcine pancreatic lipase (PPL), using thin layer chromatography to monitor the appearance of one or another hydrolysis product and to measure the rate of hydrolysis. The results of the group as a whole are then examined to assess and compare the specificity of each enzyme. In our version of the experiment each student spends 2 days in the laboratory synthesizing the diester product, then 2 days to prepare the orange peel extract and carry out the enzyme assays. Since a few of the diesters are commercially available, the enzymatic hydrolysis could be examined in isolation, if desired.
which were recrystallized. Yields were typically in the range 20–50%. Synthesis of the diester (4) was achieved by acylation of ester 2 in refluxing pyridine with the appropriate acid chloride for 1 h, followed by an aqueous workup, as described in Vogel (2, p 1248). The majority of products were isolated as liquids, which were used as isolated. Yields were typically in the range 60–90%. Purity of 4 was assessed by recording a 1H-NMR spectrum, which was subsequently assigned by the student.
Preparation of Orange Peel Extract Each student peeled the outer layer of peel (the “zest”) of one orange using a knife. The combined peel was added to 50–60 mL of 50 mM sodium citrate buffer (pH 5.5) containing 2.3% NaCl, and homogenized in a blender for 2 min until homogeneous. The blended mixture was then centrifuged at 12,000 g for 10 min, and the clear orange supernatant was decanted into a bea-
CO2R
CO2R
OCOR' OCOR' R = Me, Et, CF3CH2, iPr R' = Me, n-C5H11, n-C9H19, tBu, Ph
O R'
O O
esterase
OR O O
HO
or
R'
O O O-
+ R'CO2-
Synthesis of Diesters (see Fig. 2) Hydroxy acid (1) was converted into the hydroxy ester (2) by acid-catalyzed esterification with the appropriate alcohol (MeOH, EtOH, CF3CH2OH, i-PrOH), as described in Vogel (2, p 1077). The para- and meta-substituted products were obtained as solids, which were recrystallized, whereas the ortho-substituted hydroxyesters were obtained as liquids, which were distilled under reduced pressure. Yields were typically in the range 50–70%. Acylation of the hydroxy acid was also carried out to obtain the acylated acid (3) as a standard for the enzyme assays. Acylation was carried out by rapid treatment of 1 in aqueous sodium hydroxide with the appropriate acid chloride (CH3COCl, n-C 5H 11COCl, n-C 9H 19COCl, Me 3CCOCl, or PhCOCl) at 0 °C, as described in Vogel (2, p 1248). All products were solids,
?
OR
Experimental Procedure
*Corresponding author.
CO2R OCOR'
+ ROH
Figure 1. Structures of diester substrates used in practical experiment.
(1)
CO2H HO
O
ROH
(2)
H2SO4
HO
OR
R'COCl pyridine
R'COCl NaOH/H2O
O (3) R'
CO2H O
O
(4)
R' O
OR
O
Figure 2. Synthetic route for diester substrates.
Vol. 74 No. 1 January 1997 • Journal of Chemical Education
105
In the Laboratory
Table 1. Summary of Results of the Experimenta 1 = para-hydroxybenzoic Acid R
R'
OPE
PLE
PPL
1 = meta-hydroxybenzoic Acid OPE
PLE
1 = ortho-hydroxybenzoic Acid
PPL
OPE
PLE
PPL
Me
Me
fast(2)
fast(2)
slow(2)
fast(2)
fast(2)
med(2)
med(2;1)
fast(2)
—
Et
Me
fast(2)
fast(2)
—
fast(2)
fast(2)
slow(2)
med(2)
med(2)
slow(3)
slow(2)
med(2)
med(2+3)
slow(2)
slow(2+3)
med(3)
slow(3)
—
fast(2)
fast(2)
—
slow(2)
—
—
CF3CH2
Me
iPr
Me
Me
tBu
—
fast(2)
—
—
slow(2)
—
slow(3)
med(3)
slow(3)
Me
C5H11
fast(2)
fast(2)
med(2)
med(2)
med(2)
slow(2)
slow(3)
slow(3)
slow(3)
Me
C9H19
slow(2)
—
slow(3)
slow(2+3)
slow(2)
—
slow(3)
slow(3)
med(3;1)
Me
Ph
—
slow(2)
—
med(2)
fast(2)
—
slow(3)
slow(3)
—
med(2+3;1) med(2;1) fast(2)
fast(2)
aThe speed of enzymatic hydrolysis and the observed hydrolysis product (2 or 3) are both indicated. R,R′ are the substituents on the diester (see Fig. 1). “fast” = hydrolysis complete in 30 min; “med” = hydrolysis complete in 1–2 h; “slow” = partial/complete hydrolysis after 24 h; — = no hydrolysis; OPE = orange peel esterase; PLE = pig liver esterase; PPL = porcine pancreatic lipase. “2+3” indicates that both products 2 and 3 were observed, and “2;1” indicates that product 2 was observed, followed by hydroxy-acid 1 upon subsequent incubation.
ker for use in the enzyme assays. The protein concentration in the extract was measured using the method of Bradford (3), with bovine serum albumin as a protein standard. The extract was stored on ice and was stable for at least 24 h.
Assays for Enzyme-Catalyzed Hydrolysis The students were asked to devise a suitable thin layer chromatography system to separate their diester (4) from hydroxy-ester (2) and acylated acid (3). Typically 1:1 ethyl acetate/petroleum ether (60–80° fraction); dichloromethane; or dichloromethane/10% methanol were found to be useful eluents. The students dissolved 0.1 g of their diester (4) in 5 mL of acetone, and set up the following incubations in screw-topped vials:
hydroxy-ester 2 was commonly observed, perhaps reflecting the better leaving group properties of phenoxide vs. alkoxide. In order to encourage hydrolysis of the carboxyl ester of 4, trifluoroethyl esters were tested, and in these cases some hydrolysis to 3 was indeed observed. The ortho-substituted series showed quite varied behavior. Hydrolysis to acyl acid 3 was commonly observed, suggesting that these substrates adopt quite different orientations in the active sites of the three enzymes due to their angular substitution pattern.
PB
A. 0.1 mL diester (4) solution + 0.9 mL 50 mM sodium citrate buffer, pH 5.5 B. 0.1 mL diester (4) solution + 0.9 mL orange peel extract C. 0.1 mL diester (4) solution + 0.9 mL pig liver esterase stock (1.0 unit/mL in 50 mM potassium phosphate buffer, pH 7.0) 1 D. 0.1 mL diester (4) solution + 0.9 mL porcine pancreatic lipase stock (1.0 unit/mL in 50 mM potassium phosphate buffer, pH 7.0) 1
Ser
HS
HL
PF
O
Ser
O
The four incubations were then analyzed by thin layer chromatography at time points of 30 min, 1 h, 2 h and 24 h and the course of the enzymatic hydrolysis reaction to 2 or 3 was observed. If hydrolysis was complete after 30 min, incubations were set up with 10-fold or 100fold diluted enzyme, in order to estimate a rate of hydrolysis. Using the protein concentration of the orange peel extract and the given protein concentrations of the pig liver esterase and porcine pancreatic lipase stock solutions, rates of hydrolysis for each enzyme were calculated in units of µmol substrate min-1 mg-1 protein. The results of the group as a whole were then assessed by the students to examine the effect of changing substituents on enzyme specificity.
H3C
O O
CH3
O
Ser
O O O
Results of Experiment In all cases some enzymatic hydrolysis was observed with at least one of the three enzymes, and in nearly all cases the enzymatic hydrolysis was specific for production of either hydroxy-ester 2 or acyl acid 3. A summary of the results obtained is shown in Table 1. For the parasubstituted and meta-substituted diesters hydrolysis to
106
Figure 3. Rationalization of PLE regiospecificity for the para- and ortho- substituted diester derivatives R = Me, R′ = t-Bu, using the active site model of Jones et al. (4). HL and HS represent large and small hydrophobic binding sites, respectively, and PF and P B represent front and back polar group binding sites. The position of the active site serine nucleophile is indicated.
Journal of Chemical Education • Vol. 74 No. 1 January 1997
In the Laboratory
Of the three enzymes, PLE was generally the most active, especially for the more bulky pivalyl, hexanoyl and benzoyl esters—although it showed only weak activity with the decanoyl esters, suggesting that its active site cannot accommodate well the largest substrates. The regiospecificity observed for PLE can be rationalized by the active site model of Jones et al. (4), in which the substrate is positioned in the most favorable conformation with respect to four binding sites: large (H L) and small (HS) hydrophobic binding sites, and front (PF) and back (PB) polar group binding sites. Hydrolysis will take place if one of the two ester groups is positioned in proximity to the active site serine nucleophile (Fig. 3). For example, in the case of the pivalyl derivative (R = Me; R9 = t-Bu) hydrolysis to hydroxy-ester 2 is observed in the para-series but hydrolysis to acyl-acid 3 is observed in the ortho-series. This can be rationalized as follows: in the para-substituted substrate the aromatic ring can be positioned in the large hydrophobic (HL) site, positioning the -OCOR9 group adjacent to the active site serine and positioning the R9 substituent in the small hydrophobic (HS) site; whereas in the ortho-substituted series the aromatic ring can be positioned in the HS site, allowing the R9 group to bind in the HR site and allowing hydrolysis of the -COOR group (see Fig. 3). Orange peel esterase, for which only very limited data is available in the literature (1), was an effective catalyst for hydrolysis of acetyl and hexanoyl esters, but did not effectively accommodate more bulky side chains. PPL was generally the least active enzyme. However, the decanoyl derivative in the para-series was hydrolyzed to the corresponding acyl acid (3), suggesting that PPL
is specific for long-chain esters reminiscent of its natural lipid substrates. Thus the experiment has proved to be a successful and interesting demonstration of enzyme catalysis at the undergraduate level. The experiment is inexpensive to run and can be used for large numbers of students. The only specialized piece of equipment required is the centrifuge used to clarify the orange peel extract; presumably the experiment could be carried out using unclarified extract if necessary. Acknowledgments We would like to thank Anita Corner, John Pollard, and David Miller for technical assistance, and the 36 second-year chemistry undergraduates who enthusiastically completed the experiment in February–June 1995. Notes 1. Pig liver esterase and porcine pancreatic lipase of defined activity were purchased from Sigma Chemical Company, and were diluted into buffer as appropriate. One unit of enzyme activity is defined as the amount required to convert 1 µmol of substrate per minute.
Literature Cited 1. Kubota, Y.; Shoji, S.; Funakoshi, T.; Shionaga, K.; Ueki, H. J. Pharm. Soc. Jpn. 1983, 103, 655–661; Kubota, Y.; Shoji, S.; Funakoshi, T.; Shionaga, K.; Ueki, H. J. Pharm. Soc. Jpn. 1984, 104, 1070–1074. 2. Vogel, A. I., Vogel’s Textbook of Practical Organic Chemistry, 5th ed.; Longman, New York, 1989. 3. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. 4. Toone, E. J.; Werth, M. J.; Jones, J. B. J. Am. Chem. Soc. 1990, 112, 4946–4952.
Vol. 74 No. 1 January 1997 • Journal of Chemical Education
107
