Role of lysine ε-amino groups of β-lactoglobulin on its activating effect of Kluyveromyces lactis β-galactosidase

Article abstract of DOI:10.1021/jf703785p

Native β-lactoglobulin binds and increases the activity of Kluyveromyces lactis β-galactosidase. Construction of a three-dimensional (3D) model of β-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with β-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys¹³⁸ is the most likely to react with both. Affinity chromatography demonstrated that succinylated β-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated β-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys ε-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of β-lactoglobulin on β-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys ε-amino groups of β-lactoglobulin.

Full text of DOI:10.1021/jf703785p

J. Agric. Food Chem. 2008, 56, 5859–5863 5859
Role of Lysine ε-Amino Groups of ꢀ-Lactoglobulin on
Its Activating Effect of Kluyveromyces lactis
ꢀ-Galactosidase
†
‡
´
´
´
ELIZABETH DEL MORAL-RAMIREZ, LENIN DOMINGUEZ-RAMIREZ,
†
†
´
ALMA E. CRUZ-GUERRERO, GABRIELA M. RODRIGUEZ-SERRANO,
†
†
´
´
MARIANO GARCIA-GARIBAY, LORENA GOMEZ-RUIZ, AND
,†
´
´
JUDITH JIMENEZ-GUZMAN*
Departamento de Biotecnolog´ıa, Universidad Auto´noma Metropolitana, Iztapalapa, Mexico City,
Mexico, and Departamento de Bioqu´ımica, Laboratorio de Fisicoqu´ımica e Ingenier´ıa de Prote´ınas,
Facultad de Medicina, Universidad Nacional Auto´noma de Mexico, Mexico City, Me´xico
Native ꢀ-lactoglobulin binds and increases the activity of Kluyveromyces lactis ꢀ-galactosidase.
Construction of a three-dimensional (3D) model of ꢀ-lactoglobulin showed that lysine residues 15,
47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with ꢀ-galactosidase.
Molecular docking estimated the interaction energies of amino acid residues with either lactose or
succinic anhydride, showing that Lys138 is the most likely to react with both. Affinity chromatography
demonstrated that succinylated ꢀ-lactoglobulin diminished its ability to bind to the enzyme.
Furthermore, when activity was measured in the presence of succinylated ꢀ-lactoglobulin, its activating
effect was lost. Since succinylation specifically blocks Lys ε-amino groups, their loss very likely causes
the disappearance of the activating effect. Results show that the activating effect of ꢀ-lactoglobulin
on ꢀ-galactosidase activity is due to the interaction between both proteins and that this interaction is
very likely to occur through the Lys ε-amino groups of ꢀ-lactoglobulin.
KEYWORDS: ꢀ-Lactoglobulin; Kluyveromyces lactis ꢀ-galactosidase; Lys ε-amino groups; molecular
docking
INTRODUCTION
presence of lactose diminished its activating effect. Other reports
have established that heating ꢀ-lg in the presence of lactose
resulted in a reaction between the protein and the sugar
(lactosylation) (4), which diminished the binding capacity of
the protein to the enzyme and therefore its activating effect (3).
Jime´nez-Guzma´n et al. (3) concluded that ꢀ-lg raises ꢀ-gal
activity through two different mechanisms, one of which
depends on the release of sulfhydryl groups by heat treatment
from the denatured protein (5) and the other as a result of the
ability of the native protein to bind the enzyme (3).
KluyVeromyces lactis ꢀ-galactosidase (ꢀ-gal) is by far the
most important commercial lactase used in the dairy industry.
Several reports have established that the presence of some
proteins in the reaction medium may affect the activity of ꢀ-gal;
speculations raise the possibility that the proteins present a
masking effect of inhibiting metal ions in the reaction
medium (1, 2). Jime´nez-Guzma´n et al. (3) studied the effect of
whey proteins in ꢀ-gal activity and demonstrated that the activity
of KluyVeromyces lactis ꢀ-gal increased when it was measured
in the presence of either ꢀ-lactoglobulin (ꢀ-lg) or bovine serum
albumin; this finding is particularly interesting since these
proteins are available in milk and whey, which are the natural
reaction media for this enzyme in dairy processing. Affinity
chromatography assays demonstrated that ꢀ-gal bound specif-
ically to ꢀ-lg, resulting in enzyme activation. Moreover, heating
pure ꢀ-lg further increased ꢀ-gal activity, but heating it in the
Lysine ε-amino are some of the most exposed and reactive
groups in ꢀ-lg, with Lys47 and Lys138 being the most exposed
ones (6). It has also been reported that lactosylation of ꢀ-lg
occurs through the amino groups of lysine, specifically
Lys47 (4, 7, 8). Since it has been reported that lactosylation
causes a diminution of the interactions between ꢀ-lg and ꢀ-gal
and of its activating effect (3), it is very likely that the same
region of the molecule of ꢀ-lg may be involved in the binding
between ꢀ-lg and ꢀ-gal; thus, this interaction might be through
ꢀ-lg’s Lys ε-amino groups. The aim of this work was to
determine the role of the Lys ε-amino groups of ꢀ-lg on its
activating effect of K. lactis ꢀ-gal.
* Corresponding author. Departamento de Biotecnolog´ıa, Universidad
Auto´noma Metropolitana, Unidad Iztapalapa, Av. San Rafael Atlixco
186, Col. Vicentina, Mexico City 09340, Mexico. Tel: +(52) (55)5804-
4720. Fax: +(52) (55)5804-4712. E-mail: jjg@xanum.uam.mx.
† Universidad Auto´noma Metropolitana.
‡ Universidad Nacional Auto´noma de Mexico.
10.1021/jf703785p CCC: $40.75  2008 American Chemical Society
Published on Web 06/27/2008

5860 J. Agric. Food Chem., Vol. 56, No. 14, 2008
Del Moral-Ram´ırez et al.
MATERIALS AND METHODS
Three-Dimensional Modeling and Molecular Docking. A three-
dimensional (3D) model was built with PyMOL software (DeLano
Scientific LLC, 2007) using the NMR coordinates of bovine ꢀ-lacto-
globulin in solution obtained at pH 7.1 in deuterium and found in the
Protein Data Bank (PDB) database (PDB ID: 1CJ5) (9).
Molecular docking was then performed to locate the binding site
and estimate the binding energies between the different amino acid
residues of ꢀ-lg and lactose or succinic anhydride using AutoDock 3.05
software (The Scripps Research Institute, 2006). Results were obtained
simulating a temperature of 25 °C and were clustered taking into
account a range of rmsd of 5 Å. The 3D models for lactose and succinic
anhydride were drawn at the Dundee PRODRG2 Server site (http://
davapc1.bioch.dundee.ac.uk/programs/prodrg/).
ꢀ-Lactoglobulin Succinylation. Succinylation gradually and specif-
ically blocks the amino groups of ꢀ-lg. Succinylated ꢀ-lg (ꢀ-lgsucc) was
obtained by consecutively adding 5 mg of succinic anhydride per mg
of protein (Matheson Coleman and Bell, Ohio, USA) to 100 mL of a
5 mg/mL solution of ꢀ-lg (MP Biomedicals, Inc., Ohio, USA) and
allowing the mixture to react in constant agitation, maintaining the pH
constant at 7.0 with KOH 0.05 N (JT Baker, Xalostoc, Mexico)
according to Hollecker and Creighton (10, 11). Each time that succinic
anhydride was added, a sample was taken in order to test the effect of
different degrees of succinylation; each succinylated sample was
referred to as a derivate (D); 12 derivates (D1-D12) were obtained,
each one with a higher degree of succinylation. The remaining succinic
anhydride was eliminated by ultrafiltrating the resulting solution across
a 10 kDa cellulose membrane (Pellicon XLPLCGC 10, Millipore,
Bedford, Massachusetts, USA). To determine the degree of amino
groupsblockedinꢀ-lg,succinylationwasmonitoredthroughUrea-PAGE
(T ) 11%; c ) 0.4%; 8 M urea; pH 4.7) according to Creighton (12).
Since succinylation changes the net charge of the protein, the amount
of amino groups blocked by the reaction was determined from the
changes on the Rf of the protein bands.
Figure 1. Three-dimensional model of ꢀ-lactoglobulin constructed with
PyMOL software. Most available lysine residues are shown.
were prepared to have a concentration according to a molar ratio of
0.05 mol ꢀ-gal/mol immobilized ꢀ-lg or ꢀ-lgsucc. Immobilized ligands
and the control were allowed to interact with ꢀ-gal by directly adding
0.1 g of support with immobilized ligands to 3 mL of ꢀ-gal sample
and shaking for 1 h, then the supernatant was separated from the support
by centrifugation (3220g, 15 min) with a Beckman J2-MI centrifuge
(Beckman Instruments, Palo Alto CA, USA), and its specific activity
was measured using ONPG as a substrate.
Statistical Analyses. . Each experiment was performed three times.
Data were analyzed by means of a variance test (ANOVA); in some
cases, a Tukey test was performed. All statistical analyses were carried
out using the statistical analysis software Statistica 5.0 (Stat Soft, Tulsa
OK, USA), with p < 0.05 used as a threshold of statistical significance.
Enzyme Activity Measurement. A commercial enzyme preparation,
Maxilact LX-5000, (Gist Brocades, Delft, The Netherlands) was used
as the source of ꢀ-galactosidase. All reactions were carried out at 37
°C by dilution 1:400 of Maxilact LX-5000 into 0.05 M phosphate buffer
at pH 7.0. A solution of 0.034 M ortho-nitro-phenyl-ꢀ-D-galactoside
(ONPG) (Sigma Chemical Co.St. Louis MO, USA) was used as
substrate, and enzyme activity was measured spectrophotometrically
at 410 nm on the basis of the release of ortho-nitro-phenol (ONP) after
mixing 0.2 mL of ONPG solution with 0.1 mL of enzyme solution in
2.7 mL of phosphate buffer. The hydrolysis rate (ν0) was calculated
from the linear portion of data of the ONP production versus time.
One enzyme unit (U) was defined as the amount of enzyme that
hydrolyses 1 µmol of substrate (ONPG) in 1 min at 37 °C and pH 7.0.
Specific activity was calculated dividing by the concentration of protein
determined according to Bradford (13).
ꢀ-Gal activity of Maxilact LX-5000 was measured in the presence
of 3 mg/mL of native or succinylated ꢀ-lg. Since succinylation yields
succinic acid, pH of the succinylated derivates was neutralized with
0.05 M KOH to avoid the effect of acid pH on the activity. A control
of ꢀ-lg with 0.05 M KOH (ꢀ-lgKOH) was used to determine the effect
of the neutralizer in enzyme activity. All results were compared to a
control without protein.
Protein-Enzyme Interaction. This interaction was determined by
means of affinity chromatography using an Eupergit (Ro¨hm GmbH &
Co., Darmstadt, Germany) support with immobilized ꢀ-lg or ꢀ-lgsucc
as ligands. Immobilization was performed following the instructions
of the producer. A control without ligand was prepared by blocking
the active oxyrane groups of the support with glycine. After im-
mobilization, the amount of immobilized protein was calculated through
the difference between the protein in solution before and after interacting
with the support. Because of the difference in the available lysines of
native and succinylated ꢀ-lg, the efficiency of immobilization was
RESULTS AND DISCUSSION
Reactivity of ꢀ-Lactoglobulin’s Lys ε-Amino Groups.
Previous reports have established that the lactosylation of ꢀ-lg
diminishes the protein’s ability to activate ꢀ-gal, probably
because of the loss of its capability to bind to the enzyme (3);
hence, it has been suggested that the same region involved in
the binding of lactose could be the one involved in the activation
of the enzyme. It has also been reported that ꢀ-lg can react with
lactose through the first stages of the Maillard reaction (lacto-
sylation) (4). In order to evaluate the role of the amino groups
of ꢀ-lg in the activation of the enzyme, molecular modeling
and molecular docking were used to study the interactions
between ꢀ-lg and lactose.
A 3D model of ꢀ-lg was built on the basis of the NMR
coordinates of the protein in solution (9) in order to study the
exposition and possible steric hindrance that the different lysines
in the molecule would show when reacting with lactose and/or
another protein. Taking into account that pH could affect the
exposition of the different amino acids, the model was con-
structed at pH 7.1, which is the optimum pH of K. lactis ꢀ-gal
and the pH at which all other experiments were performed.
ꢀ-Lg has one free amino group and other 15 lysine residues
in its molecule, each one with different reactivity. Upon
analyzing our 3D model of ꢀ-lg (Figure 1), it was observed
that Lys47 and Lys138 are very exposed in the molecule, and
lysine residues 15 and 69 are also exposed at this pH, suggesting
that any of these amino acids could be very reactive.
There are some controversies about which lysines are the most
reactive lysines in ꢀ-lg. Several authors have reported that Lys47
is one of the most exposed and reactive groups in the
molecule (4, 7, 8) and that this is in fact the first amino acid
participating in the lactosylation reaction, while others also
different in the cases of both molecules (0.5374 and 0.3157 µmol/gsupport
,
respectively).
Maxilact LX-5000, determined by electrophoresis to contain a
mixture of eight proteins, was used as the ꢀ-gal sample. Because of
the differences in the amount of protein immobilized, ꢀ-gal samples

Role of Lysine of ꢀ-Lactoglobulin on ꢀ-Galactosidase Activity
J. Agric. Food Chem., Vol. 56, No. 14, 2008 5861
Figure 3. Molecular docking of succinic anhydride (gray and red) and
ꢀ-lactoglobulin showing succinic anhydride bound to Lys138
Figure 2. Molecular docking of lactose (gray and red) and ꢀ-lactoglobulin
.
showing lactose bound to Lys138
.
consider Lys138 as one of the most reactive ones (6). Fogliano
et al. (14), however, reported Lys100 as the most reactive one;
they also suggested that the reactivity of the different Lys
ε-amino groups in the protein depends on the conditions of the
medium in which it is dissolved. Our results show that even
though there are several lysine residues in the outer face of the
protein, at pH 7.1 the most exposed ones are Lys47 and Lys138
,
which are the ones more likely to interact with another protein.
Molecular docking of lactose and ꢀ-lg (Figure 2) showed
that the amino acid with most favorable interaction energy at
pH 7.1 was Lys138, which suggests that the most probable site
of interaction between the protein and the sugar is this amino
acid. The docking program used was blind, which means that
it had no previous information about which amino acids are
known to react with the sugar, and therefore, it calculates the
interaction energies with all the amino acid residues of the
protein. Hence, it was remarkable that among all the amino acid
residues, lysines were the ones with lowest interaction energies
ranging from -8.2 to -4.0 kcalmol-1, and among those, Lys138
showed the highest probability to be the binding site with a
∆G binding of -4.7 kcal mol-1. This indicates that lysine residues
easily react with lactose through a nucleophilic attack and that
their exposure at different conditions facilitates or inhibits this
interaction.
Jime´nez-Guzma´n et al. (3) reported that when ꢀ-lg is
lactosylated its interaction with ꢀ-gal diminishes and so does
its activating effect on the enzyme; nevertheless, it is not known
whether the interaction is diminished because of the steric
hindrance by lactose inhibiting the protein’s interaction through
different amino acid residues of the same region or if the amino
groups of ꢀ-lg that are blocked by lactose are in fact important
for the protein-enzyme interaction. Because of this, succinic
anhydride was chosen as an electrophile to react with ꢀ-lg; this
is a very small molecule, which as long as the protein is not
denatured, would not cause steric hindrance for any other groups
to interact with ꢀ-gal.
Figure 4. Activity of ꢀ-galactosidase in the presence of either ꢀ-lg or its
different succinylated derivates. Control refers to the activity measured in
buffer solution without protein; N refers to native ꢀ-lg; ꢀ-lgKOH refers to
the neutralized native ꢀ-lg solution; D1-D12 refer to the different
succinylated derivates of ꢀ-lg.
its native conformation at least until half the amino groups have
been succinylated (11). In order to determine if amino groups
of lysine actively participate in the activation of the enzyme,
ꢀ-gal activity was measured in the presence of either native or
blocked ꢀ-lg and compared with a control without protein; since
succinylation yields succinic acid, pH of the succinylated
derivates was neutralized using KOH, and a control of ꢀ-lg with
KOH (ꢀ-lgKOH) was also used to determine the effect of the
neutralizer on the activity.
The presence of native ꢀ-lg or ꢀ-lgKOH (Figure 4) increased
the hydrolysis rate with respect to the control in about 147% (p
< 0.001), confirming the activating effect of ꢀ-lg reported by
Jime´nez-Guzma´n et al. (3); the activity measured in the presence
of ꢀ-lgKOH was not significantly different (p > 0.1) from that
of native ꢀ-lg, suggesting that KOH had no effect on enzyme
activity. When activity was measured in the presence of the
first succinylated derivates, the activating effect was still
observed (Figure 4, bars D1-D2). Nevertheless, in the presence
of the rest of the succinylated derivates, activity decreased
reaching the same value of the control without protein, showing
no significant difference with it (p > 0.05) (Figure 4, bars
D4-D12).
To determine the degree of amino groups blocked in the
different succinylated derivates, succinylation of ꢀ-lg was
monitored through 8 M Urea-PAGE. According to Hollecker
and Creighton (11), succinylation of each amino group causes
a change in the migration of the protein (Rf) due to the change
in the protein’s charge. When analyzing the Rf values of the
different derivatives (Figure 5), it was observed that from D1
to D12, the amount of amino groups blocked varied from one
to five. Furthermore, results showed that in the first two
succinylated derivates even though a band of succinylated ꢀ-lg
with one Lys ε-amino group blocked can be observed (Figure
In order to support that succinic anhydride reacts with the
same amino acids as lactose, molecular docking of succinic
anhydride and ꢀ-lg was done (Figure 3). Results showed that
Lys138 was the only amino acid with favorable interaction energy
at pH 7.0 (∆G
) -6.6 kcal mol-1). This is the same
binding
amino acid interacting with lactose, suggesting that succinic
anhydride could be a small molecule that simulates the reaction
with lactose without causing steric hindrance. Hence, succinic
anhydride was used as a model to study the effect of blocking
the amino groups of ꢀ-lg on ꢀ-gal activity.
Effect of Succinylation of Amino Groups of ꢀ-Lactoglo-
bulin on ꢀ-Galactosidase Activity. Succinylation gradually and
specifically blocks the amino groups of ꢀ-lg without changing

5862 J. Agric. Food Chem., Vol. 56, No. 14, 2008
Del Moral-Ram´ırez et al.
Figure 6. Specific activity of ꢀ-galactosidase after affinity chromatography
using ꢀ-lg or succinylated ꢀ-lg as ligands showing the differences in the
binding of ꢀ-galactosidase by the proteins. Initial refers to the specific
activity of the solution before affinity chromatography. Control refers to
the specific activity obtained when no ligand was bound to the support.
Figure 5. Urea-PAGE electrophoretic pattern of either native ꢀ-lg (N)
or its different succinylated derivates (D1-D12). The numbers on the
left side specify the number of lysine ε-amino groups that are blocked in
each derivate.
ꢀ-lg as ligand, the specific activity of the supernatant decreased
significantly (70% ( 1.3, p < 0.01) with respect to the control
(support without ligand) and the original solution, showing that
ꢀ-gal bound specifically to ꢀ-lg. When ꢀ-lgsucc was used as
ligand, even though some of the protein in Maxilact LX-5000
was bound to the column, the specific activity of the supernatant
was not significantly different from that of the control (p >
0.1) (Figure 6). This can be explained by the unspecific
adsorption of the protein in the resin, but there is no evidence
of a specific interaction with ꢀ-gal.
The fact that no interaction and no activation are detected
when Lys ε-amino groups of ꢀ-lg are blocked suggests that these
groups could be involved in the protein’s ability to bind to the
enzyme and hence its capability to activate ꢀ-gal. The interaction
of the protein with ꢀ-gal could induce some structural changes
of the enzyme, and according to Tello-Sol´ıs et al. (15), small
conformational changes (in that case induced by pH) can give
up important activation of K. lactis ꢀ-galactosidase.
Consequently, it has been clearly demonstrated that blocking
Lys ε-amino groups diminishes the interactions between ꢀ-lg
and ꢀ-gal, thus causing the loss of its activating effect. It is
clear that amino groups from lysine are essential for this effect;
nevertheless, at this point it is not possible to define whether
this effect is due to a single lysine residue such as Lys138 or
Lys47, or to more than one of them. Further work is being done
to clarify this issue.
5, D1-D2), native ꢀ-lg was still present at high concentrations,
while in the rest of the succinylated derivates (Figure 5,
D4-D12), native ꢀ-lg was not observed. Moreover, D2 and
D4 contain two, and two and three lysine amino groups blocked,
respectively, and the main difference found between them was
the presence of native ꢀ-lg in D2. When comparing with the
activation results, it seems clear that while native ꢀ-lg was still
present in the reaction medium, enzyme activation was observed
(D1 and D2), but when all of ꢀ-lg is succinylated in at least
one amino group (D4-D12), the activating effect was lost. At
this point, the most reactive amino groups of ꢀ-lg were blocked
and stable, suggesting that the loss in activating capacity is due
to the blocking of its amino groups.
Even though in the first succinylated derivatives some amino
groups are already blocked, the activating effect is still present.
This could be due either to the fact that even small amounts of
ꢀ-lg are able to activate the enzyme or to the fact that more
than one Lys ε-amino is involved in the interaction with the
enzyme.
Effect of Succinylation on the Interaction of ꢀ-Lactoglo-
bulin with ꢀ-Galactosidase. Jime´nez-Guzma´n et al. (3) dem-
onstrated that the activating effect of ꢀ-lg could be due to the
interaction of ꢀ-lg with ꢀ-gal. In order to study the effect of
Lys ε-amino groups in the interaction of ꢀ-lg with ꢀ-gal, the
capacity of ꢀ-lgsucc (the equivalent to D12) to bind to ꢀ-gal was
compared with that of native ꢀ-lg through affinity chromatog-
raphy, immobilizing either ꢀ-lg or ꢀ-lgsucc on Eupergit and
allowing the immobilized ligands to interact with a solution of
Maxilact LX-5000. A control was used in which no protein was
immobilized, but the reactive oxyran groups of the resin were
blocked with glycine.
Specific activity relates the activity ratio to the amount of
protein present in a given sample and therefore helps to establish
the proportion of enzyme among other contaminating proteins.
When the interaction with the ligand is unspecific, any of the
proteins can be bound to the support, but their proportions in
the eluting mixture will not change; however, when there is a
specific interaction between one protein (in this case ꢀ-gal) and
the ligand, its proportion in the eluting mixture will diminish.
Results showed that the specific activity of the control was not
significantly different from that of the initial ꢀ-gal solution (p
> 0.1) meaning that ꢀ-gal was not specifically bound to the
support without protein (Figure 6). When the same solution of
ꢀ-gal (Maxilact LX-5000), containing eight proteins visible by
electrophoresis, was mixed with the support containing native
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