UDP-galactose 4-epimerase from Kluyveromyces
fragilis – catalytic sites of the homodimeric enzyme
are functional and regulated
Amrita Brahma*, Nupur Banerjee* and Debasish Bhattacharyya
Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology (CSIR), Jadavpur, Kolkata, India
Introduction
UDP-galactose 4-epimerase, hereafter called epimerase,
is an essential and ubiquitous enzyme that reversibly
converts UDP-Gal to UDP-Glc. The epimerase from
the yeast Kluyveromyces fragilis is a homodimer of
nearly 75 kDa per subunit, and contains bound
NAD
+
acting as cofactor [1–3]. Epimerases from Esc-
herichia coli [4–6], Saccharomyces cerevisiae [7] and
human sources [8] have been cloned and sequenced,
and their X-ray crystallographic structures are known.
The bacterial enzyme has two NAD
+
-binding sites
Keywords
catalytic sites; inhibitor; multimeric enzyme;
regulation; UDP-galactose 4-epimerase
Correspondence
D. Bhattacharyya, Structural Biology and
Bioinformatics Division, Indian Institute of
Chemical Biology (CSIR), 4, Raja S. C.
Mallick Road, Jadavpur, Kolkata 700 032,
India
Fax: +91 33 2473 5197 ⁄ 0284
Tel: +91 33 2499 5764

enzyme after treatment with p-chloromercuribenzoate indicated stability of
the dimeric enzyme against fast association–dissociation, which could
otherwise generate multiple forms of the enzyme with functional
heterogeneity.
Abbreviations
CHD, 1,2-cyclohexanedione; GG, glycylglycine; pCMB, p-chloromercuribenzoate; STI, soybean trypsin inhibitor.
FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6725
away from the subunit contact region, and its mono-
mers are functional [9].
The molecular mass of the yeast enzyme is almost
double that of the bacterial and human epimerases. A
blast search of the yeast epimerase revealed two fea-
tures: its N-terminal half showed strong homology
with the E. coli epimerase, and the C-terminal half
showed homology with mutarotase [10]. The predicted
mutarotase activity in K. fragilis epimerase was later
demonstrated [11,12]. Furthermore, the enzyme can be
cleaved by trypsin into two parts in the presence of
epimerase and mutarotase inhibitors. They can func-
tion independently as an epimerase and a mutarotase
[11]. Interestingly, when trypsin digestion is performed
in the presence of only the epimerase inhibitor, the
mutarotase domain is fragmented, yielding a 45 kDa
monomeric epimerase [11,13].
The yeast enzyme exhibits a stoichiometry of two
NAD
+
molecules per dimer, similarly to E. coli and
human epimerase, raising the possibility of the exis-
tence of two catalytic sites. Binding of one molecule of

4
.E
1
, epimerase containing one 5¢ -UMP per dimer bound as isolated (native epimer-
ase); [E
2
], an intermediate of the conversion where the unoccupied 5¢-UMP-binding site of E
1
is occupied by the added 5¢-UMP (the bracket
indicates its transient character); E
3
, stable intermediate where the 5¢-UMP bound ex vivo to E
1
is replaced allosterically by the added
5¢-UMP; E
4
, epimerase where both the 5¢-UMP binding sites are occupied by added 5¢-UMP; [E
2A
], product of reductive inhibition of [E
2
] with
L(+)-arabinose (the bracket indicates uncertainty about its existence); E
3A
, product of reductive inhibition of [E
3
] with L(+)-arabinose. The two
lobes in all the structures indicate homodimeric epimerase; the flange at the middle of each lobe separates the epimerase (upper) and
mutarotase (lower) domains of a monomer; the rectangular denting of the upper domains of each lobe indicates the binding site of 5¢-UMP
ex vivo; the shaded rectangle indicates 5¢-UMP bound as isolated; the open rectangle indicates added 5¢-UMP;
•

as free nucleotide or bound to other enzymes
is not sensitive to this reaction), whereas the inhibitor
bound in vitro does [14].
Here, we provide a hypothesis for the pathway fol-
lowed by epimerase as isolated (E
1
) during its satura-
tion with extraneously added 0.5 mm 5¢-UMP (E
4
)
(Scheme 1). An essential feature in this proposal is that
the nature of the binding of 5¢-UMP in E
1
and that in
E
4
are different, evidence for which has been men-
tioned above. On the basis of the situation in
Scheme 1, the minimum requirement for the conver-
sion is the existence of two intermediates, E
2
and E
3
.
In E
1
, one inhibitor-binding site is occupied and the
other is vacant. In E
2
, the added 5¢-UMP binds to the

3
should be independent of added 5¢-UMP. A cor-
ollary of the prediction is that half of the catalytic sites
of E
1
and E
3
remain bound to 5¢-UMP, whereas both
of the catalytic sites in E
2
and E
4
are occupied by the
inhibitor. Thus, after incubation with trypsin, the
residual activities of E
1
,E
2
(if it exists), E
3
and E
4
are
expected to be 0%, 50%, 50% and 100%, respectively,
on the basis of selective protection by the added inhib-
itor. Similarly, the formation of NADH and residual
activity after reductive inhibition in the presence of
only l(+)-arabinose for these enzyme–inhibitor com-
plexes are expected to be 0, 1, 1 and 2 mol per dimer
and 100%, 50%, 50% and 0%, respectively. To be

Conversion of NAD
+
to NADH by reductive inhibi-
tion of epimerase offered a sensitive method for deter-
mination of stoichiometry of the bound cofactor.
Reductive inhibition was applied to 33.3 nmol (5 mg)
of epimerase. A control set of the enzyme under identi-
cal conditions but in the absence of the reducing
agents [5¢-UMP and l(+)-arabinose] retained 98% of
the activity. Complete inactivation of the enzyme
ensured quantitative reduction of NAD
+
to NADH.
Dissociation of NADH from the enzyme was achieved
with 8 m urea [17], and was quantified spectrofluori-
metrically. Recovery of NADH was 63.0 ± 4.0 nmol,
yielding a stoichiometry of 1.89 ± 0.12 per dimer, or
close to 1.0 NADH per subunit (n = 4). Further
improvement in quantification was restricted by the
uncertainty of protein concentration determination and
the incompleteness of conversion of NAD
+
to NADH.
Therefore, with respect to the composition of cofactor,
the catalytic sites of epimerase remain indistinguish-
able.
Stability of subunits
According to Scheme 1, the subunits of epimerase (E
1
)

remained unchanged when the enzyme was incubated
in the assay mixture without the substrate for 30 min
at 25 °C prior to activity measurements. These obser-
vations collectively indicate that epimerase does not
undergo rapid exchange of subunits during catalysis.
Characterization of epimerase–inhibitor
complexes
The equilibrium intermediates formed during conver-
sion of the inhibitor complex E
1
to E
4
in the presence
of 0–0.6 mm 5¢-UMP were characterized by a kinetic
lag in catalysis, ‘coenzyme fluorescence’ (described
later), and inactivation by trypsin (Fig. 2A), and two
other parameters of reductive inhibition, namely inacti-
vation and formation of NADH (Fig. 2B). The last
two parameters are sensitive enough to be measured
with an accuracy of ± 0.25%.
The dependency of the lag in catalysis followed a
sharp decrease of 100% to 10% ± 2% in the presence
of 0–0.2 mm 5¢-UMP. This indicated removal of the
inhibitor bound as isolated in E
1
or modification of
the inhibitor-binding site. A corresponding conforma-
tional change at the cofactor-binding site was moni-
tored from coenzyme fluorescence. There was an initial
25% rise in emission intensity in the presence of

stituted epimerase. Units of the ordinate and abscissa are lmolÆ-
min
)1
and mM
)1
, respectively; values correspond to the original
plot.
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6728 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS
completed with 0.5 mm 5¢-UMP, as higher concentra-
tions of the inhibitor failed to cause additional change
(Fig. 2A).
Proteolysis of native epimerase by trypsin is initiated
from an arginine residue located at the catalytic site
[11]. The degradation can be prevented either by modi-
fication of this residue by CHD or by incubation with
5mm 5¢-UMP [16]. 5¢-UMP can protect E
4
against
trypsin, but not E
1
. Furthermore, as the arginine at
the catalytic site of E
1
does not appear to be protected
by 5¢-UMP, the amino acid could be modified by
CHD, which in turn is expected to resist trypsin diges-
tion. SDS ⁄ PAGE of CHD-modified E
1
showed genera-

with a stable intermediate in the presence of 0.1–
0.2 mm 5¢-UMP. Formation of E
3A
from E
3
represents
the reductive inhibition of the stable intermediate. This
intermediate possesses residual activity of 58% ± 2%
as compared with E
1
, and NADH fluorescence of
63% ± 2% as compared with E
4
. The NADH fluores-
cence was measured under denaturing conditions to
remove interference from coenzyme fluorescence. This
indicated that under the defined conditions of reduc-
tive inhibition, one of the two NAD
+
molecules of the
dimeric enzyme was converted to NADH. In control
experiments, it was verified that the reagents carried
over to the assay mixture did not cause inactivation of
the coupled enzyme. Therefore, the results of
Fig. 2A,B are in agreement with Scheme 1.
Irreversible conversion
If the conversion of Scheme 1 were reversible, the
enzyme–inhibitor complexes would become unstable
while the excess inhibitor was removed. The complexes
E

FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6729
sodium phosphate (pH 7.5) at 4 °C to remove
unbound inhibitor. It was verified that the native epim-
erase (E
1
) could withstand inactivation due to dialysis
under such conditions. The presence of 5¢-UMP in all
of the dialyzed samples was confirmed by MS analysis
(Fig. 3). The dialyzed enzyme complexes were sub-
jected to reductive inhibition in the presence of 10 mm
l(+)-arabinose. The residual activities of E
1
,E
3
and
E
4
were 80% ± 5%, 55% ± 5% and 8% ± 5%,
respectively (n = 2). The kinetic lag in catalysis as
observed with E
1
could not be reproduced with the
dialyzed samples of E
3
and E
4.
This indicated that the
inhibitor-binding steps are irreversible.
Change in tertiary structure
A change in conformation of an enzyme is an obligatory

), and mono-
meric epimerase (E
M
), were used for kinetic analysis in
the presence of 0–0.35 mm substrate (Figs 5–7).
Fig. 3. MS analysis of the dissociated ligand of native epimerase. The observed peaks have been assigned as follows: 5¢-UMP, 2Na
+
,
H
+
= 369.1 (obs. 368.99); 5¢-UMP, 2Na
+
,2H
+
= 370.1 (obs. 369.87); 5¢-UMP, 3Na
+
= 391.1 (obs. 390.96); and 5¢-UMP, 3Na
+
,H
+
= 392.1
(obs. 391.87). Commercially available 5¢-UMP-disodium salt, under similar experimental conditions, showed an identical mass pattern. The
spectral zone of NAD
+
has not been included.
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6730 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS
Regulation of catalytic activity has been clearly dem-
onstrated in the case of the native epimerase. The
Michaelis–Menten relationship showed hyperbolic

emission intensity of native epimerase is considered to be 100% in
either set. The ligands had no emission in this spectral zone.
AB
Fig. 6. Michaelis–Menten plot of inhibitor-free epimerase with
UDP-Gal as substrate. The enzyme concentration was 1.65 n
M. The
open circles (s—s) indicate experimentally observed points. (A, B)
Lineweaver–Burk plots with 0–0.05 m
M and 0.05–0.35 mM sub-
strate. Units of the ordinate and abscissa are lmolÆmin
)1
and
m
M
)1
, respectively; values correspond to the original plot. Derived
values of K
m
and V
max
are presented in Table 1. The line (
•
—
•
)
was constructed according to Eqn (2), using the parameters of
Table 1. The other line is the best fit joining the experimentally
observed points (s—s).
Fig. 7. Michaelis–Menten plot of monomeric epimerase with
UDP-Gal as substrate. Inset: Lineweaver–Burk plot with 0–0.35 m

)1
for the high-affinity site, and 1.0 mm and
5.56 lmolÆmin
)1
Æmg
)1
for the low-affinity site, respec-
tively. The presence of 5¢-UMP bound to the enzyme
as isolated was detected from the kinetic lag in cataly-
sis and the characteristic MS pattern. When these anal-
yses were performed, the enzyme was incubated with
variable concentrations of the substrate for 1 min
under the assay conditions, and was passed through a
spin column to separate unbound ligands from the
eluted enzyme. It was observed that, in the presence of
up to 0.06 mm UDP-Gal, the inhibitor remained
bound to the enzyme (Fig. 5, solid and hatched bars).
Thus, the catalytic site of the native epimerase, which
was free of the inhibitor, was nonfunctional at low
substrate concentrations.
In the case of inhibitor-free epimerase, the depen-
dency of reaction velocity on substrate concentration
cannot be represented by a single Michaelis–Menten
relationship over the range of substrate concentrations
used, because the corresponding Lineweaver–Burk plot
had a poor correlation (R
2
= 0.8473, where R
2
is the

meric epimerase showed hyperbolic dependency and a
linear Lineweaver–Burk plot between 0 mm and
0.35 mm UDP-Gal. Derived K
m
and V
max
values were
0.01 mm and 2.52 lmolÆmin
)1
Æmg
)1
, respectively, sug-
gesting that the catalytic site was similar to the high-
affinity site of the native and inhibitor-free epimerase
(Fig. 7 and inset). All kinetic parameters are summa-
rized in Table 1.
Assessment of kinetic data
When the epimerase reaction did not show a Michaelis–
Menten relationship, it was assumed that the cata-
lytic sites were operating simultaneously at unequal
efficiencies. Under such conditions, the rate of an
enzyme reaction (V) can be expressed as the sum of two
Michaelis–Menten dependencies, as in Eqn (2) [21].
V ¼
V
max ðHÞ
½S
K
m ðHÞ
þ½S

from
V
max
.
From the values of K
m
and V
max
(Table 1), the
dependency of V on [S] was calculated between 0 mm
and 0.35 mm UDP-Gal and compared with the experi-
mental data. For inhibitor-free epimerase, the correla-
tion was quite satisfactory (R
2
= 0.982) (Fig. 6). In
the case of native epimerase, Eqn (2) was not expected
to be valid, as the catalytic sites were not operating
simultaneously (Fig. 5). Analysis of Fig. 5 showed that
contributions by the high-affinity and low-affinity sites
to overall turnover were 34.2% and 65.8%, respec-
tively, when maximum turnover by the enzyme was
achieved. This is in agreement with the profiles of
Fig. 2A,B, where inactivation of one catalytic site by
trypsinization or reductive inhibition led to residual
activities of 61.3% and 59.9%, respectively. Deviation
from equal catalytic efficiency of the two functional
Table 1. Kinetic properties of different forms of epimerase. The
high-affinity and low-affinity sites refer to the substrate UDP-Gal.
Results shown are within ± 5% error.
Epimerase

catalytic sites at infinite substrate concentration
reached 100%, although these values are different in
absolute terms because of regulation. The analysis
shows that raising the substrate concentration from
0.001 mm to 0.025 mm increased the activities of the
high-affinity and low-affinity sites from 0.90% to
69.4% and from 0.06% to 12.26%, respectively. When
the substrate concentration was further increased from
0.025 mm to 0.35 mm, the corresponding increases
were 69.4–96.96% and 12.26–66.27%, respectively.
Effects of inhibitor
The range of inhibitor concentrations and the pattern
of dependency of inhibition of regulatory enzymes dif-
fer from those of Michaelis–Menten-type enzymes [21].
Competitive inhibition of epimerase by 5¢-UMP is
known [14–16,20,22]. Typical plots of residual activities
of native and monomeric epimerase versus inhibitor
concentration show that the profiles are widely differ-
ent (Fig. 8). In the case of native epimerase, no inhibi-
tion was observed up to 0.8 mm 5¢-UMP, as compared
with 62.5% inhibition for the monomeric epimerase.
At 20 mm 5¢-UMP, the monomeric epimerase showed
76.3% inhibition, the native epimerase showed 85%
inhibition. Dixon plots (inverse of rate versus inhibitor
concentration) of the monomeric and native epimerase
were hyperbolic and parabolic (results not shown). The
hyperbolic dependency indicated partial inhibition
from a single binding site of the inhibitor in mono-
meric epimerase. The parabolic dependency indicated
two binding sites of the inhibitor in native epimerase

at 4 °C for 40 h at pH 7.5. Whereas native epimerase
without any reagent retained 96% ± 2% of its activ-
ity, incubation with any combination of reagents
reduced the activity to 65% ± 5%, with a distinctly
different pattern in the Michaelis–Menten plot. There
was a hyperbolic dependency between 0 mm and
0.075 mm UDP-Gal, after which there was no signifi-
cant change in the catalytic rates up to 0.35 mm sub-
strate. This evidently indicates inactivation of the
second site (Fig. 9A). Inhibitor-free epimerase incu-
bated with various combinations of reagents demon-
strated the same biphasic Michaelis–Menten
dependency as that shown in Fig. 6, with 80% ± 3%
recovery of residual activity (Fig. 9B).
Discussion
Allosteric regulation of the epimerase from K. fragilis
has not been investigated with confidence before. That
there is deviation from the Michaelis–Menten relation-
ship during the reversible conversion of UDP-Gal to
Fig. 8. Inhibitory profiles of native and monomeric epimerase by
5¢-UMP. The enzyme and substrate concentrations were 1.65 n
M
and 0.1 mM, respectively. 5¢-UMP has no effect on the coupling
enzyme. The enzyme activity in the absence of the inhibitor is
considered to be 100%.
A. Brahma et al. Regulation of catalytic sites of yeast epimerase
FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6733
UDP-Glc before product equilibration is attained has
been known for a long time [24]. The allosteric kinetics
of the epimerase were reported more recently [25,26].

The resulting solution had no characteristic NADH
fluorescence to the limit of detection (< 0.01 mol per
dimer). Incomplete recruitment of NAD
+
and reacti-
vation of the enzyme during the assay after it has
absorbed NAD
+
from the assay mixture can also
cause functional heterogeneity. This was ruled out, as
the enzyme preincubated with 0.05 mm NAD
+
for
15 min prior to the assay did not show enhancement
of activity. Reductive inhibition of epimerase followed
by quantification of dissociated NADH (as illustrated
in Experimental procedures) showed that the stoichi-
ometry was nearly 2.0 per dimer or 1 per catalytic site.
Earlier, maximum recovery of 1.70 ± 0.10 mol of
NAD per dimer was reported, based on dissociation of
the nucleotide by trichloroacetic acid or heat, where
partial coprecipitation of the holoenzyme with the
apoenzyme is suspected [14].
The stability of dimeric structure of epimerase with
regard to rapid association–dissociation was estab-
lished from complete and reversible dissociation of the
subunits after modification with pCMB, followed by
reduction under nondenaturing conditions [19]. The
kinetic parameters of the reconstituted enzyme were
distinctly different from those of the native enzyme

, epimerase incu-
bated without any substrate analog.
, enzyme preincubated
with 0.2 m
M UDP + 2 mMD(+)-Gal. , enzyme preincubated
with 0.2 m
M UDP + 2 mMD(+)-Glc. (B) Inhibitor-free epimerase.
•
—
•
, dependencies of inhibitor-free epimerase preincubated
without substrate analogs.
, dependencies of inhibitor-free
epimerase preincubated with 0.2 m
M UDP + 2 mMD(+)-Gal.
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6734 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS
yielded a residual activity of 58% ± 2% (n = 6). This
indicates that the catalytic sites of the enzyme are
functional and are distinguishable on the basis of bind-
ing with the inhibitor. Careful analysis of Fig. 2 shows
that inactivation of E
3
by trypsin and reductive inhibi-
tion consistently deviated from 50%, in spite of modi-
fication of one catalytic site of two. Such inequality
between two identical catalytic sites is usually achieved
by allosteric regulation. A perceivable change in the
conformation of the enzyme in the presence of
5¢-UMP and UDP-Gal was demonstrated by trypto-

cies. The nonidentical nature of the Michaelis–Menten
relationships of inhibitor-free and native epimerase is
an indication of the regulatory role of the bound
5¢-UMP in the latter. The Michaelis–Menten relation-
ship of the monomeric epimerase displays the usual
monophasic hyperbolic dependency, which further vali-
dates cooperativity between the two catalytic sites
(Fig. 7). In this context, we reviewed why previously
reported K
m
values varied between 0.1 mm and
0.13 mm [15,16,24]. In those cases, a double reciprocal
plot of the native epimerase was constructed between
0.025 mm and 0.5 mm UDP-Gal, a 20-fold variation in
concentration, but without attention being paid to
even lower concentrations of the substrate. The exis-
tence of inhibitor-free epimerase was unknown at that
time.
The deviation from the Michaelis–Menten relation-
ship, including sigmoidal kinetics, is a necessary but
not sufficient condition for allostericity. Several fac-
tors, including impurities from reagents or the presence
of interfering enzymes, could alter the enzyme kinetics
[28]. The monophasic Michaelis–Menten relationship
of the monomeric epimerase demonstrated the absence
of such artefacts and the requirement for a dimeric
structure to explain the regulation. The difference in
V
max
values among different forms of the epimerase is

them.
Biological significance
It is pertinent to ask why the native epimerase is the
only isolable form of the enzyme from yeast cells har-
vested near termination of growth. The content of
inhibitor-free epimerase is gradually reduced with the
concomitant rise of the native form in a time-depen-
A. Brahma et al. Regulation of catalytic sites of yeast epimerase
FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6735
dent manner from initiation to termination of cell
growth [14]. In vitro experiments show that inhibitor-
free epimerase cannot be reversibly converted to the
native form. It therefore appears that, at a late phase
of cell growth, the enzyme remains inactive after bind-
ing to substoichiometric amounts of 5¢-UMP. On the
other hand, the concentration of free nucleotides in
microbial cells is 25–50 nmolÆmg
)1
of protein [29]. This
concentration is 100-fold lower than that applied to
convert the inhibitor-free epimerase to the native form.
Consequently, spontaneous conversion of native epim-
erase to E
3
or E
4
in vivo is a remote possibility. This
consideration is important in maintaining the
UDP-Gal or UDP-Glc pool in the cell, as well as in
ascertaining the homogeneity of native epimerase in its

merase that is devoid of 5¢-UMP; monomeric epimerase
or E
M
, a 45 kDa truncated and monomeric functional
form of native epimerase that is devoid of the C-terminal
mutarotase domain. The notations E
1
–E
4
have been used in
the context of binding of the inhibitor 5¢-UMP to epimerase.
The terms native, inhibitor-free and monomeric epimerase
have been used in the context of kinetic properties of the
enzyme.
Reagents
d(+)-Gal, d(+)-Glc, l(+)-arabinose, glycylglycine (GG),
CHD, b-NAD
+
, UDP-Gal, UDP-Glc, 5¢-UMP, UDP,
UTP, DTT, pCMB, soybean trypsin inhibitor (STI), trypsin
(bovine pancreas) and hydroxyapatite were from Sigma
(USA). Urea (GR) was recrystallized from hot ethanol. The
yeast K. fragilis (ATCC 10022, renamed Kluyveromy-
ces marxianus var. marxianus) was purchased from the
Microbial Type Collection Center and Gene Bank, IM-
TECH, Chandigarh, India. YNB (yeast nitrogen base) was
from Hi-media, Mumbai, India. UDP-Glc dehydrogenase
(EC 1.1.1.22) was partially purified from beef liver up to
the heat denaturation step [32]. This enzyme was left for
15 days at ) 20 °Cin50mm sodium acetate (pH 5.5),

)
Epimerase (0.5 mgÆmL
)1
in 50 mm GG, pH 8.8) was trea-
ted with 1 mm UDP-Gal for 15 min at 25 °C, whereby the
bound inhibitor was replaced by the substrate [14]. Excess
substrate and inhibitor were removed by passage through a
Sephadex G-50 spin column. MS analysis showed that this
enzyme was devoid of 5¢-UMP.
Preparation of monomeric epimerase (E
M
)
Epimerase (1 mgÆmL
)1
) was treated with trypsin (50 : 1,
w ⁄ w) in 20 mm potassium phosphate (pH 8.0) at 4 °C for
4 h in the presence of 0.5 mm 5¢-UMP. Residual trypsin
was inactivated by the addition of a two-fold molar excess
of STI. The digest was passed through a Waters Protein
Pak 125 size exclusion HPLC column equilibrated with
20 mm sodium phosphate (pH 7.5) at a flow rate of
0.5 mLÆmin
)1
, and the major peak, corresponding to
45 kDa, was collected. Alternatively, 100 lL of the digest
was passed through a Sephadex G-50 spin column to
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6736 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS
remove reagents and small peptides. The recovery of mono-
meric epimerase from the spin column was 95% in terms of

Glc dehydrogenase in 1 mL. It was incubated for 10 min at
25 °C to remove contaminating UDP-Glc present in the
UDP-Gal. Unless preincubation was performed, an initial
burst phase was observed as an artefact. The assay was ini-
tiated by adding 3–15 nmol of the epimerase. The rate of
formation of product showed linear dependency for at least
8 min, when the absorbance change per minute was within
0.03. The rate of product formation also showed linear
dependency when the epimerase concentration was
3–15 nm. In that profile, the extrapolated line passed
through the origin, indicating that the coupling enzyme was
free from epimerase activity. By varying the volume of
enzyme added between 10 lL and 400 lL instead of water
in the assay mixture and observing the linear progress curve
of D0.005–0.03 absorption unitsÆmin
)1
, the assay permits
detection of as little as 0.25% of enzyme activity. To study
the effects of inhibitor, the assay mixture was incubated
with 0–20 mm 5¢-UMP for 10 min at 25 °C; the reaction
was initiated by adding the epimerase. During the epimer-
ase assay, the UDP-Gal concentration could not be raised
above 0.5 mm, where the coupling enzyme became limiting.
The coupling enzyme UDP-Glc dehydrogenase was assayed
with UDP-Glc as substrate in the presence of NAD
+
at
340 nm, under the same conditions as those used for the
epimerase assay [15]. The points presented in all kinetic
experiments (Figs 4–9) are the average of three sets, where

M
, monomeric epimerase where
there is only one 5¢-UMP-binding site occu-
pied by added 5¢-UMP and the mutarotase
part is degraded. E
0
, dimeric epimerase
where both the catalytic sites are occupied
by UDP-Gal (also referred to as inhibitor-free
epimerase). The solid rectangle indicates
added UDP-Gal bound to the epimerase; s,
arginine at the active site; s over UDP-Gal
binding site indicates protection against
trypsinization in the presence of UDP-Gal.
All other notations are as in Scheme 1.
A. Brahma et al. Regulation of catalytic sites of yeast epimerase
FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6737
whereby the dimeric enzyme was dissociated with release of
the cofactors [19]. It was passed through a Sephadex G-50
spin column to remove excess reagents and free NAD
+
.
Reconstitution of the dimeric holoenzyme was initiated by
adding 50 mm dithiothreitol and 1 mm NAD
+
, and
90–100% reactivation was achieved by 30 min at 25 °C.
Coenzyme fluorescence
Yeast epimerase possesses an inherent NADH-like charac-
teristic fluorescence (excitation, 353 nm; emission, 400–

Quantitative conversion of epimerase bound NAD
+
to
NADH was performed by reductive inhibition. Completion
of the reaction was indicated by complete inactivation of
the enzyme. The reduced cofactor was dissociated from the
enzyme after incubation with 8 m urea at pH 7.5 for
10 min. The concentration of the reduced cofactor was
determined from the fluorescence intensity with respect to a
calibration curve. The curve correlated the fluorescence
intensity of NADH in the presence of 8 m urea with its
concentration (0–10 lm), where a linear dependency was
observed (R
2
= 0.9983). The presence of globular proteins
without visible chromophore did not interfere with NADH
emission. In all fluorescence experiments, the inner filter
effect was allowed for [17,34,35].
Trypsin digestion
Digestion of epimerase by trypsin was performed essentially
as described for preparation of monomeric epimerase,
except for the variation in 5 ¢-UMP concentration. Unbound
5¢-UMP and small peptides so formed were separated
from the undigested epimerase by passage through a
Sephadex G-50 spin column. The functional enzyme was
recovered from the eluent for assay.
Characterization of epimerase–inhibitor
complexes
Epimerase (0.25–0.5 mgÆmL
)1

ine of the enzyme, leading to NADH-like coenzyme fluorescence. The subscript ‘free’ denotes dissociated nucleotides. Enzyme-bound and
free NADH fluorescence are similar but not identical.
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6738 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS
been ensured, the NADH fluorescence of incubates was
measured. It was verified that none of the reagents used in
these experiments interfered with NADH fluorescence.
MS
A Q-TOF micro (Micromass) instrument with microchannel
plate detectors was used. Positive ionization electrospray
mode at a desolvation temperature of 200 °C was applied.
Argon, at a pressure of 2 kgÆcm
)2
, with a collision energy
of 10 eV, was used as collision gas. Epimerase
(0.05 mgÆmL
)1
) was dialyzed extensively against water at
4 °C, lyophilized, and reconstituted in 10 mm potassium
phosphate (pH 7.0) at a concentration of 0.5 mgÆmL
)1
. The
sample was heated at 100 °C for 5 min to dissociate 5¢-
UMP and NAD
+
. The solution was centrifuged at 5585 g
for 15 min to remove precipitated protein. The components
in the supernatant were separated by RP-HPLC before
mass analysis. 5¢-UMP and NAD
+

incubated with its substrate analogs, which were combina-
tions of 0.25 mm uridine nucleotides (UMP, UDP, or UTP)
and 2 mm reducing sugars [d(+)-Gal, d(+)-Glc, or l(+)-
arabinose] in 0.05 m potassium phosphate (pH 7.5) at 4 °C
for 40 h. As a positive control, an enzyme without any sub-
strate analog was incubated under identical conditions,
where 2–3% of inactivation was observed. After incubation,
the rates of enzymatic conversion were measured in the
presence of 0–0.35 mm UDP-Gal.
Other methods
A UV–visible recording spectrophotometer (Specord 200;
Analytical Jena, Germany) was used for enzyme assays.
Other optical measurements were performed with a Bio-
chrom S2000 diode array spectrophotometer (UK). All flu-
orescence measurements were performed with a
Hitachi F4500 recording spectrofluorimeter, using 700 lL
quartz cuvettes with excitation and emission slit widths of
5 nm each. Arginines of epimerase (0.5 mgÆmL
)1
) were
modified with 2 mm CHD in 0.2 m sodium borate (pH 9.0)
for 3 h at 37 °C [3]. Protein estimation was performed
according to the method of Lowry et al. [37] or with
Bio-Rad Protein Assay Reagent, as per the manufacturer’s
protocol (catalog no. 10044; Bio-Rad Laboratories), using
BSA as reference. The following extinction coefficient val-
ues were used: NADH, e
340 nm
= 6.3 · 10
3

Department of Science and Technology (Grant
No. SR ⁄ SO ⁄ BB-66 ⁄ 2005) awarded to D. Bhattachar-
yya. A. Brahma and N. Banerjee were supported by
CSIR-SRF and UGC NET-SRF, respectively.
References
1 Frey PA (1987) Complex pyridine nucleotide-dependent
transformations. In Pyridine nucleotide coenzymes:
chemical, biochemical and medical aspects (Dolphin D,
Poulson R & Avarmovie O, eds), Vol. 2B, pp. 461–511.
Wiley, New York, NY.
2 Bhattacharjee H & Bhaduri A (1992) Distinct functional
roles of two active site thiols in UDP-galactose 4-epim-
erase from Kluyveromyces fragilis. J Biol Chem 267,
11714–11720.
3 Frey PA (1996) The Leloir pathway: a mechanistic
imperative for three enzymes to change the stereochemi-
cal configuration of a single carbon in galactose.
FASEB J 10, 461–470.
4 Thoden JB, Frey PA & Holden HM (1996) High-resolu-
tion X-ray structure of UDP-galactose 4-epimerase
complexed with UDP-phenol. Protein Sci 5, 2149–2161.
5 Thoden JB, Frey PA & Holden HM (1996) Crystal
structure of oxidized and reduced forms of UDP-galac-
tose 4-epimerase isolated from E. coli. Biochemistry 35,
2557–2566.
6 Thoden JB & Holden HM (1998) Dramatic differences
in the binding of UDP-glucose and UDP-galactose to
UDP-galactose 4-epimerase from Escherichia coli.
Biochemistry 37, 11469–11477.
7 Thoden JB & Holden HM (2005) The molecular

14 Nayar S, Brahma A, Barat B & Bhattacharyya D
(2004) UDP-galactose 4-epimerase from Kluyveromy-
ces fragilis: analysis of its hysteretic behavior during
catalysis. Biochemistry 43, 10212–10223.
15 Darrow RA & Rodstrom R (1968) Purification and
properties of uridine diphosphate galactose 4-epimerase
from yeast. Biochemistry 7, 1645–1654.
16 Mukherjee S & Bhaduri A (1986) UDP-glucose 4-epim-
erase from Saccharomyces fragilis: presence of an essen-
tial arginine residue at the substrate-binding site of the
enzyme. J Biol Chem 261, 4519–4524.
17 Bhattacharyya D (1993) Reversible folding of UDP-
galactose 4-epimerase from yeast Kluyveromyces fragilis.
Biochemistry 32, 9726–9734.
18 Darrow RA & Rodstrom R (1970) Uridine diphosphate
galactose 4-epimerase from yeast: studies on the rela-
tionship between quaternary structure and catalytic
activity. J Biol Chem 245, 2036–2042.
19 Majumdar S, Bhattacharjee H, Bhattacharyya D &
Bhaduri A (1998) UDP-galactose 4-epimerase from
Kluyveromyces fragilis: reconstitution of holoenzyme
structure after dissociation with para-
chloromercuribenzoate. Eur J Biochem 257, 427–433.
20 Roy S, Mukherjee S & Bhaduri A (1995) Two trypto-
phans at the active site of UDP-glucose 4-epimerase
from Kluyveromyces fragilis . J Biol Chem 270, 11383–
11390.
21 Roberts DV (1977) Regulatory Enzymes and their
Kinetic Behavior.InEnzyme Kinetics. pp 168–227.
Cambridge University Press, Cambridge.

30 Citri N (1973) Conformational adaptability in enzymes.
Adv Enzymol Relat Areas Mol Biol 37, 397–648.
31 Traut TW (1994) Dissociation of enzyme oligomers: a
mechanism for allosteric regulation. Crit Rev Biochem
Mol Biol 29 , 125–163.
32 Zalitis J, Uram M, Bowser AM & Feingold DS (1972)
UDP-glucose dehydrogenase from beef liver. Methods
Enzymol 28, 430–435.
33 Nath S, Brahma A & Bhattacharyya D (2003) Extended
application of gel-permeation chromatography by spin
column. Anal Biochem 320, 199–206.
34 Dutta S, Maity NR & Bhattacharyya D (1997) Multiple
unfolded states of UDP-galactose 4-epimerase from
yeast K. fragilis: involvement of proline cis–trans isom-
erization in reactivation. Biochim Biophys Acta 1343,
251–262.
35 Maiti NR, Barat B & Bhattacharyya D (1999) UDP-
glucose 4-epimerase from Kluyveromyces fragilis: equi-
librium unfolding studies. Indian J Biochem Biophys 36,
433–441.
36 Kalckar HM, Bertland AU & Bagge B (1970) The
reductive inactivation of UDP-galactose 4-epimerase
from yeast and E. coli. Proc Natl Acad Sci USA 65,
1113–1119.
37 Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the folin-phenol
reagent. J Biol Chem 193, 265–276.
Regulation of catalytic sites of yeast epimerase A. Brahma et al.
6740 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS