Carbohydrate Polymers 102 (2014) 615–621

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

A novel catalysis by porcine pepsin in debranching guar

galactomannan
Mysore S. Shobha a , Lalitha R. Gowda b , Rudrapatam N. Tharanathan a,∗
a
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570 020,
India
b
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570
020, India

a r t i c l e i n f o a b s t r a c t

Article history: Background: Pepsin (porcine stomach mucosa, E.C. 3.4.23.1), an acid protease catalyzes the hydrolysis
Received 9 April 2013 (debranching) of guar galactomannan (GG), a co-polymer of mannose and galactose residues thereby
Received in revised form showing its non-specific catalysis towards glycosidic substrates.
16 November 2013
Results and conclusions: Use of non-specific inhibitors, chemical modification agents and peptide mapping
Accepted 27 November 2013
of native and GG – bound pepsin upon proteolytic digestion with Staphylococcus aureus V8 protease
Available online 4 December 2013
revealed the involvement of Asp138 residue in the catalysis, which was confirmed by computational
modelling studies.
Keywords:
Porcine pepsin
General significance: Here we show a novel mode of catalysis (other than proteolysis) by porcine pepsin
Guar galactomannan with a different active site residue.
Non-specificity © 2013 Elsevier Ltd. All rights reserved.
Peptide mapping
Active site
Docking

1. Introduction and characterized three chitosanase isozymes from porcine pepsin,

and showed that 14 out of first 15 N-terminal amino acid residues
GG, a branched co-polymer of mannose and galactose residues, of isozyme PSC-III were identical with those of pepsin B.
obtained from the seeds of leguminous plant Cyamopsis tetrago- In this study, an attempt has been made to unravel why and
nalobus finds numerous applications in various food and non-food how pepsin – a proteolytic enzyme brings about catalysis of struc-
industries, and therefore is of high commercial importance turally unrelated carbohydrate substrates. The use of site specific
(Greenberg & Slavin, 2003; Kokol, 2002). High branching, high vis- inhibitors, chemical modification agents and peptide mapping of
cosity and high molecular weight of GG, nevertheless, restricts native and GG-bound pepsin on proteolytic digestion with Glu-C
additional uses of GG, which was partly overcome by selective V8 protease were carried out to know the binding site residue and
removal of side chain galactose residues using ␣-galactosidase. also to elucidate the mode of action of pepsin in debranching of GG.
Though successful, the utility of this technology is restricted Pepsin, a monomeric ␤-protein (Mr , 34.6 kDa) with two domains
because of the laborious steps involved and cost effectiveness comprising a high percentage of acidic residues (43 out of 327 are
for isolating the pure enzyme. A few earlier reports claiming aspartic and glutamic acids, pKa – 1.5), of which Asp32 (ionized) and
the multiple specificity of several enzymes on various carbo- Asp215 (unionized) are mainly responsible for proteolysis. Though,
hydrate macromolecules (Vishu kumar, Gowda, & Tharanathan, functionally a proteolytic enzyme, the non-specific reactivity of
2004; Pantaleon, Yalpani, & Scollar, 1992; Shobha, Vishu Kumar, pepsin towards glycosidic substrates may be due to structural fea-
Tharanathan, Rathna, & Gaonkar, 2005), emerged as an alternative tures mimicking several carbohydrate degrading enzymes, which
approach for the application of non-specific enzymes in modifying are characterized by the presence of two domains with barrel-like
guar galactomannan. Fu, Wu, Chang, and Sung (2003) also isolated structures having one end of the barrel wider than the other. At the
wider end is an elongated cleft, which would become narrower or
wider depending on the size of the substrate (Jedrzejas, 2000). The
∗ Corresponding author. Tel.: +91 080 23198195. pairwise sequence alignment of porcine pepsin (5 pep) with guar
E-mail address: tharanathan@yahoo.co.uk (R.N. Tharanathan). ␣-galactosidase showed 25% sequence homology (Fig. 1), whose

0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.11.043
616 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621

Fig. 1. Multiple alignment of porcine pepsin and guar ␣-galactosidase.

active sites contain an Asp residue involved in the catalysis of their acetic acid–water (5:2:5). Destaining was carried out using an aque-
cognate substrates. ous solution of 30% methanol–10% acetic acid.

2. Materials and methods 2.4.2. Activity towards GG

After electrophoresis the gel was incubated with 0.5% GG at 40 ◦ C
2.1. Materials for 8 h in acetate buffer (0.1 M) of pH 5.5 and later stained with 0.1%
Congo red (1 h), excess stain was removed using 1 M potassium
Pepsin (porcine stomach mucosa, E.C. 3.4.23.1), haemoglobin chloride.
(Bovine), pepstatin, phenylmethylsulfonylketone (PMSF), N-tosyl-
l-lysine-chloromethylketone hydrochloride (TLCK), iodoacetate, 2.5. MALDI-TOF-MS
N-(3-methyl amino propyl) N -ethylcarbodiimide hydrochloride
(EDAC), glycine methyl ester (GME), endoproteinase Glu-C (Staphy- The purified pepsin was mixed with an equal volume of matrix
lococcus aureus strain V8, E.C. 3.4.21.19) and Sephadex G-100 were (␣-cyano-4-hydroxycinnamic acid prepared in CH3 CN:H2 O:TFA,
from Sigma Chemicals Co., St. Louis, MO, USA. Guar gum was a gift 80:20:0.1), dried at 25 ◦ C under atmospheric pressure and trans-
from Hindustan Gum Chemicals, Haryana, India. All other reagents ferred into the vacuum chamber of the mass spectrometer
and chemicals were of highest purity available. (Compact Analytical SEQ MALDI-TOF-MS, Kratos, UK) in a reflective
positive ion mode.
2.2. Gel permeation chromatography (GPC)
2.6. Automated gas-phase protein sequencing
The crude enzyme solution was loaded onto Sephadex G-100
column (100 cm length x 0.7 cm i.d.) and eluted with 50 mM acetate The N-terminal sequencing of purified pepsin was carried out on
buffer of pH 5.6. Fractions, analyzed at 280 nm, which showed Applied Biosystems 491A automated gas phase protein sequencer
maximum proteolytic activity and also activity towards GG were (Procise 491A) by Edman degradation using the standard protocols.
pooled.
2.7. Enzyme assay
2.3. Native/SDS PAGE
Proteolytic activity of purified pepsin was assayed spectropho-
Native and SDS-PAGE was done on 7.5% gel according to the metrically using a Shimadzu UV-Visible spectrophotometer at
method of Laemmli (1970) and the isolated protein bands were 280 nm, by incubating with haemoglobin (2.5%) at pH 1.5–2.0,
visualized by staining with Coomassie Brilliant Blue (CBB). for 1 h, 37 ◦ C, and estimating the TCA-soluble peptides released.
Specific activity was expressed as one unit = absorbance at
2.4. Zymogram analysis 280 nm/reaction time × mg protein in the reaction mixture.
Non-specific hydrolytic activity was assayed using GG (0.5%,
2.4.1. Proteolytic activity w/v) solution containing pure pepsin (100 ␮g), incubated for 1 h at
In addition to the various analytical methods reported else- 37 ◦ C (Shobha & Tharanathan, 2008). The reducing sugar, released
where, the zymogram analysis of pepsin towards specific substrate into the supernatant was estimated by the ferricyanide method
was carried out according to Lopez, Lopez, Lopez, Carreno, and Toro (Imoto & Yagishita, 1971). One unit of activity was expressed as
(1998). After electrophoresis (acid PAGE), the gel was immersed ␮moles of reducing equivalents released min−1 mg−1 protein.
in 0.1 M HCl to reduce the pH to 2.0. After 15 min, the gel was
immersed in a solution containing 0.25% haemoglobin in 0.1 M 2.8. Galactose to mannose ratio (G/M)
glycine–HCl, pH 2.0 at 4 ◦ C for 30 min, then for 90 min in a fresh
haemoglobin solution at 37 ◦ C. The gel was washed with distilled The G/M ratio of pepsin treated GG was evaluated, after acid
water and fixed for 15 min in 12% trichloroacetic acid (TCA), and hydrolysis followed by derivatization into alditol acetate, by GC on
then stained with 0.1% Coomassie brilliant blue in methanol–glacial OV-225 (3% on Chromosorb W) column connected to Shimadzu 8A
 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621 617

equipped with flame ionization detector (Sawardekar, Slonekar, & and its sequence alignment with that of ␣-galactosidase was car-
Jeanes, 1965). ried out using CLUSTAL X (Higgins, Thompson, & Gidson, 1996) with
The hydrolysis of GG was further studied in the concentrated subsequent gap adjustments. The model for porcine pepsin was
supernatant of enzyme digest by HPLC on a precalibrated (using derived from the co-ordinates of the structure labelled 5pep in the
galactose and mannose, 10 ␮L) ␮-Bonda pack aminopropyl col- RCSB Protein Data Bank, which represents pepsin at 2.3 Å resolu-
umn (4.1 mm × 300 mm) using acetonitrile:water (75:25) mixture tion (Cooper, Khan, Taylor, Tickle, & Blundell, 1990). The ligand GG
as mobile phase at a flow rate of 1 mL min−1 and RI Detector. (with 6 residues) was constructed and submitted to the PRODRG
site (Schuettelkopf & van Aalten, 2004). The initial geometry and
2.9. Fluorescence studies topologies were retrieved. The ligand was docked against porcine
using Arguslab (Thompson, Planaria software) with a grid spacing
Fluorescence measurements were recorded on a Shimadzu RF of 0.4 Å. The best docked calculations and graphical manipulations
5000 spectrofluorimeter using 5 nm path length at 27 ◦ C. Fluo- were performed using pyMol.
resence emission spectra were recorded (300–450 nm) with the
excitation wavelength set at 280 nm. Appropriate blanks were used
for baseline correction of fluorescence intensity. For fluorimet- 3. Results and discussion
ric titration, 3.0 mL of solution with appropriate concentrations of
pepsin (100 ␮g mL−1 ) were titrated in acetate buffer (0.1 M) of pH 3.1. Criteria of homogeneity
5.5 by successive addition of GG solution (0–0.5%).
As the commercial enzyme preparations usually are admixed
2.10. Inhibitory studies with inorganic substances, used as enzyme stabilizers, attempts
were made to purify them before undertaking further detailed
The inhibitory effects of pepstatin, PMSF, TLCK and iodoac- structural studies, in order to rule out the involvement of these
etate were carried out by pre-incubating purified pepsin with the contaminants in non-specificity.
inhibitors at varying concentration (2–10 mM) for 1 h at 45 ◦ C. The The commercial preparation of porcine pepsin on native PAGE
residual protease activity and activity towards GG were assayed. (Fig. 2A) showed a major protein band along with the presence
of a few minor bands. Thus in order to rule out the possible
2.11. Chemical modification of carboxyl residues contribution of these minor bands for their non-specific action
towards GG the enzyme preparation was purified by preliminary
Carboxyl residue modifications were performed at 25 ◦ C with size exclusion chromatography. The protein fraction that exhib-
pepsin in 0.05 M sodium acetate (pH 4.8), containing 0.275 M GME ited maximum catalytic activity towards both protein and GG was
and 0–0.3 M EDAC (Hoare & Koshland, 1967). EDAC and GME pooled, which upon native and SDS-PAGE showed a single pro-
were dissolved in water immediately before use and inactiva- tein of Mr ∼ 34,900 ± 1200 Da (determined by standard molecular
tion initiated by the addition of EDAC. A control experiment of weight markers) corresponding to the earlier major protein band
enzymes and the nucleophile GME in buffer was run simulta- of the crude pepsin, thus ruling out the involvement of contam-
neously, which corresponds to 100% pepsin activity towards GG. inants in catalytic action (Fig. 2A). Capillary zone electrophoresis
Aliquots at 10 min intervals were removed for residual activity (CZE) and Rp-HPLC profiles also established the existence of a
determination. The pseudo first order rate constants for the inacti- single protein. The N-terminal sequence of the purified protein
vation were determined by a plot of log% residual activity against by Edman degradation revealed Ile-Gly-Asp-Glu-Pro-Leu-Glu-Asn-
time. The inactivation kinetics was fitted to the equation: log (% Tyr-Leu-Asp-Thr-Glu-Tyr-Phe which was identical to the sequence
residual activity) = −ki t, where ki is the pseudo first-order inacti- of porcine pepsin reported earlier (Sepulveda, 1973). MALDI-TOF-
vation rate constant for a given concentration of EDAC and t is time MS also revealed a single peak of Mr 34,511 Da (Fig. 2B), which
of inactivation. The inactivation order (n) was calculated from the was in concordance with the reported molecular weight of porcine
equation: log Ki = n log[inactivator] + Ki , where log Ki is the second- pepsin. These results, taken together established the homogeneity
order inactivation constant. The stoichiometry of the inactivation of the enzyme beyond ambiguity. The zymogram analysis of the
reaction was determined by a plot of log Ki versus log(EDAC). The purified pepsin on acid-PAGE for proteolytic activity and activity
slope of the curve represents the stoichiometry. towards GG showed a single clearing zone confirming the dual role
of pepsin in both proteolysis and debranching of GG (Fig. 3).
2.12. Peptide mapping of GG binding site of pepsin The effect of varying pepsin concentration was directly propor-
tional to the increased rate of catalysis with first order reaction
Native and GG-bound pepsin were incubated with endopro- kinetics upto a concentration of 100 ␮g, following zero order, and
teinase Glu-C from S. aureus strain V8 (100:3, w/w) in 0.1 M hence for further study, an enzyme:substrate ratio of 1:100 was
phosphate buffer, pH 7.8 for 18 h at 37 ◦ C. The proteolytic diges- employed. The enzyme was stable up to 5 h by retaining 90–95%
tion was arrested by heating the reaction mixture for 20 min in a activity, showed maximum stability at pH 5.5 and 45 ◦ C with
boiling water bath. The digest was concentrated to dryness and re- Km and Vmax values of 5.0 mg mL−1 and 2790 nmoles min−1 mg−1 ,
dissolved in 0.5 mL of 0.1% TFA (Matsudaira, 1989). The peptides respectively (Table 1). Various authors (Roncal, Oviedo, Lopez de
released were then separated by Rp-HPLC using a C-18 column Armentia, Fernandez, & Villaran, 2007; Vishu Kumar, Varadaraj, &
(4.6 mm × 150 mm; 5 ␮m) on a Shimadzu LC-10 system equipped Tharanathan, 2007) also report the hydrolysis of chitosan by pepsin
with a binary pump using Solvent A (0.1% TFA in water) and sol- at pH 4.5 and 5.0 respectively. Schlamowitz and Peterson (1959)
vent B (70% CH3 CN containing 0.05% TFA) (Mahoney & Hermodson, suggest the pH dependent broad specificity for the action of pepsin
1980). at a flow rate of 0.7 mL min−1 with a linear increase in the on proteins at mildly acidic range (i.e. pH 4 to higher). Campos and
solvent strength from 0 to 100% in 85 min (Hermodson & Mahoney, Sancho (2003) states pepsin (pH 4–6.5) still retaining its conforma-
1983). The peptides were detected at 230 nm. The peptide pro- tion and substrate binding site similar to its original conformation
files were compared and analyzed with those derived from native at lower pH. Further reports from Monu, Prasanna Kumari, Vikash
pepsin. A distinct peptide was collected and its sequence deter- Kumar, Pinky, and Jagannadham (2009) and Kamatari, Dobson, and
mined by Edman degradation. The amino acid sequence of porcine Konno (2003) also substantiate the native conformation of pepsin at
pepsin was retrieved from Swiss-Port/TrEMBL (Acc.No. P00791) pH 5.6 by NMR and CD measurements. Thus, though the enzyme at
618 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621

Fig. 2. (A) Native PAGE of crude pepsin (Lane 1) and purified pepsin (Lane 2); SDS PAGE of purified pepsin (Lane 3) and protein markers (Lane 4) (B) MALDI-TOF-MS of
purified pepsin.

this pH range shows changes in the proteolytic activity, the overall

insignificant changes in the structure and other biophysical prop-
erties might favour the enzyme binding to another substrate like
carbohydrate, which has made this enzyme to have broad sub-
strate specificity. The pH dependent broad specificity of pepsin at
mildly acidic pH probably may lead to the expansion of protein
allowing the compact active site to stretch a bit due to non-specific
long range interaction between different protonated groups (Goto
& Fink, 1989). Such a changes at acidic pH from a largely unfolded
state to an intermediate conformational state have been reported
for cytochrome C (Ohgushi & Wada, 1983) and human interferon
␥ (Arakawa, Hsu, & Yphantis, 1987). Fluorescence titration of GG
with pepsin resulted in an increased quenching as the concentra-
tion of the former increased, suggesting the hydrolytic action of the
pepsin.
The GLC analysis of the pepsin treated GG revealed a G:M ratio of
1:3.6 almost similar to LBG (data not shown). Thus the change in the
G:M ratio agreed well with the action of pepsin towards GG. Also the Fig. 3. Zymogram analysis of pepsin on (A) acid PAGE for proteolytic and (B) GG.
HPLC characterization of the supernatant showing the presence of
galactose alone further supported the debranching action of pepsin
on GG.
TLCK had no effect on both the activities, thereby ruling out the
contribution of serine, cysteine and histidine residues in the catal-
3.2. Identification of GG binding site of pepsin ysis of GG. All these results are suggestive of a different carboxyl
group, other than that required for proteolysis, in the cataly-
Pepstatin (3 mM), an inhibitor specific to aspartate proteases, sis of GG. It is well documented that polysaccharide degrading
totally inhibited the proteolytic activity without altering the activ- enzymes exhibit acid–base catalytic mechanism, comprising a pair
ity towards GG. Thereby suggesting different active site residues of carboxyl residues, of which one is protonated and the other
involved in hydrolysis of GG by pepsin. The addition of PMSF and unprotonated.

Table 1
Kinetic parameters of pepsin towards GG.

Enzyme pH optimum Temperature optimum (◦ C) Km (mg mL−1 ) Vmax (nmoles min−1 mg−1 protein)

Pepsin 5.5 45 5.0 ± 0.088 2790 ± 32.8

 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621 619

Fig. 4. Inactivation of pepsin by EDAC and GME. (A) Plot of log (% residual activity) versus time. (B) Plot of pseudo first order inactivation rate constant as a function of EDAC
concentration. (C) Double logarithimic plot of pseudo first order inactivation rate constant as a function of EDAC concentration.

Incubation of pepsin with either GME or EDAC, specific for modi- 3.3. Docking of GG with pepsin
fication of carboxylic groups resulted in no inhibition of the enzyme
activity (Fig. 4). However the requirement of a relatively high con- Docking simulations use computational methodologies to
centration of EDAC for the inactivation is probably due to the high mimic the biochemical process of a ligand binding in protein-ligand
content of carboxylic groups in pepsin. A similar requirement has interaction studies. Fig. 6 shows computational simulation model
been reported for various enzymes (Kanade, Paul, Appu rao, & of GG interacting with pepsin, which revealed Asp138 to be involved
Gowda, 2006; Nyvall, Pedersen, Kenne, & Gacesa, 2000). The semi- in catalyzing the removal of galactose residues. The GG–pepsin
logarithmic plot of residual activity at various EDAC concentrations complex showed a mean binding energy of −6.5 kcal mol−1 with
versus time was linear indicating that the inactivation followed Asp138 residue at a distance of 6.4 Å from the glycosidic oxygen
pseudo first-order kinetics. A plot of the first-order inactivation rate of galactose residue. Also, the imidazole of Pro126 and the phenyl
constant (ki ) against EDAC concentration was also linear. A plot of ring of Phe141 of pepsin showed a perfect stacking with galactose
log ki versus log[EDAC] yielded a slope of 1.0613. Since the stoi- and mannose of GG, thereby facilitating effective binding. Liu, Lai,
chiometry was near to 1.0 with respect to EDAC, it suggested the Chou, Chang, and Lyu (2007) report the hydrophobic interaction
involvement of a single carboxylate group in the catalytic activity of aromatic side chain of Trp and Tyr with the glucose ring of ␤-
of pepsin towards GG. cyclodextrin influencing the surrounding residues in the binding
In order to identify the GG binding site, the purified pepsin, sub- of ligand. The contribution of Asp142 (identified from GG-binding
jected to proteolysis with Glu-C V8 protease in the presence and peptide sequence) in debranching of GG is also evident from its
absence of GG, was analyzed by Rp-HPLC (Fig. 5A and B). A distinct close proximity to the substrate in the ligand bound complex. Gen-
peak with a retention time of 27.5 min was detected in the peptide erally acid catalysis by hydrolases follows a double displacement
profile of pepsin digested with the enzyme in the presence of GG. mechanism which requires two acidic residues in the vicinity of the
The sequence of this peptide, as determined by Edman degradation active site leading to hydrolysis of the substrate. Nevertheless, an
was NH2 -NLWDQGLVSQ corresponding to the residues 139–148 alternative mechanism with a single carboxylate residue has also
of porcine pepsin. In other words, the binding of GG with pepsin been reported. Weaver, Grutter, and Matthews (1995) discount the
protected the Asp142 residue from proteolysis, which substanti- need of a second carboxylate group (Asp52 ) for catalysis by goose
ated the loss of enzyme activity upon chemical modification with egg white lysozyme thereby ruling out the double displacement
EDAC. mechanism. Perrakis (1994) and Terwisscha van Scheltinga, Lalk,
620 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621

Fig. 5. Rp-HPLC profile of the proteolytic digests of pepsin in the absence (A) and presence (B) of GG.

Beintema, and Dijkstra (1994) report the X-ray crystal structure of water thereby pushing forward the reaction and also reprotonat-
family 18 chitinases with no second acidic residue in the active site ing Asp138 . Bjelic and Aqvist (2004) report a similar mechanism
required to stabilize the oxocarbonium ion. involving the direct participation of single catalytic aspartate per-
Based on the above results, a probable mechanism to explain the forming both acid–base functions, with the neighbouring histidine
hydrolytic activity of pepsin towards GG has been proposed. Amist residue providing necessary stabilization along the reaction. The
a nonpolar milieu contributed by Val136 , Phe137 and Leu140 , the pro- docking simulations of GG with pepsin showed a distance of 6.4 Å
tonated Asp138 donates a proton to the glycosidic oxygen between between the Asp138 and glycosidic oxygen and 10.4 Å between
the C-1 of galactose and C-6 of mannose residue (␣-1,6 linkage), Asp142 and oxocarbonium ion, which is greater than the normally
which results in the cleavage of the glycosidic bond thereby cre- required 3–4 Å. This increased distance may possibly explain the
ating a positive charge on the C-1 of galactose ring resulting in a lower activity towards GG when compared to its proteolytic activ-
transient oxocarbonium ion, that is stabilized by the neighbouring ity. It is also evident that such an increased distance contributes
negatively charged Asp142 , which is in a polar environment. The to the slowing down the rate of hydrolysis (Brameld & Goddard,
oxocarbonium ion intermediate reacts with incoming OH− of 1998).
 M.S. Shobha et al. / Carbohydrate Polymers 102 (2014) 615–621 621

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