Rate enhancements brought about by uridine nucleotides in the reduction of NAD+ at the active site of UDP-galactose 4-epimerase

Article abstract of DOI:10.1006/bioo.1999.1158

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glucose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible oxidation to the 4-keto- glucopyranosyl ring by reaction with the enzyme-bound NAD⁺. The UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the enzyme that enhances the chemical reactivity of NAD⁺ toward reducing agents. This is modeled by UMP-dependent reductive inactivation of the epimerase-NAD⁺ complex by various sugars as well as by borohydrides. The present work shows that UDP also activates the reduction of epimerase-bound NAD⁺. Furthermore, the reduction of epimerase-NAD⁺ by glucose at a very slow rate can be observed under anaerobic conditions in the absence of a uridine nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD⁺ by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of the rate enhancements brought about by UMP and UDP to be estimated. The rate enhancements by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol^-1, respectively, in the activation energy. A decrease of 4.0 kcal mol^-1 in the activation energy for reduction by NaBH₃CN was brought about by UMP-binding. The maximum increases in the reduction potential of epimerase-NAD⁺ induced by UMP- and UDP-binding are estimated to be 120 and 90 mV, respectively. The results are well correlated with the perturbations of the nicotinamide-¹³C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220-12230). (C) 2000 Academic Press.

Full text of DOI:10.1006/bioo.1999.1158

Bioorganic Chemistry 8, 29–37 (2000)
doi:10.1006/bioo.1999.1158, available online at http://www.idealibrary.com on
Rate Enhancements Brought About by Uridine Nucleotides
+
in the Reduction of NAD at the Active Site
of UDP-Galactose 4-Epimerase
Yijeng Liu, Abolfazl Arabshahi, and Perry A. Frey
Institute for Enzyme Research, The Graduate School, and Department of Biochemistry, College of
Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705
Received July 12, 1999
UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glu-
cose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible
+
oxidation to the 4-keto-glucopyranosyl ring by reaction with the enzyme-bound NAD . The
UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the
+
enzyme that enhances the chemical reactivity of NAD toward reducing agents. This is mod-
+
eled by UMP-dependent reductive inactivation of the epimerase-NAD complex by various
sugars as well as by borohydrides. The present work shows that UDP also activates the reduc-
+
+
tion of epimerase-bound NAD . Furthermore, the reduction of epimerase-NAD by glucose at
a very slow rate can be observed under anaerobic conditions in the absence of a uridine
+
nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD
by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of
the rate enhancements brought about by UMP and UDP to be estimated. The rate enhance-
−
1
ments by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol , respectively, in
−
1
the activation energy. A decrease of 4.0 kcal mol in the activation energy for reduction by
NaBH CN was brought about by UMP-binding. The maximum increases in the reduction
potential of epimerase-NAD induced by UMP- and UDP-binding are estimated to be 120 and
0 mV, respectively. The results are well correlated with the perturbations of the nicoti-
3
+
9
1
3
namide- C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey,
P. A. (1993) Biochemistry 32, 13220–12230).
© 2000 Academic Press
1
+
UDP-galactose 4-epimerase from Escherichia coli is an NAD -containing en-
zyme that catalyzes the interconversion of UDP-glucose and UDP-galactose by a
+
dehydrogenation mechanism, in which enzyme-bound NAD is reversibly reduced
by either UDP-galactose or UDP-glucose, with the intermediate production of a UDP-
4
-ketohexopyranose (1). The uridine nucleotide moiety of substrates is postulated to
+
activate the enzyme by increasing the reactivity of the epimerase-bound NAD toward
reduction (1,2). The occurrence of this activation is supported by several lines of evi-
1
+
Abbreviations used: UMP, uridine 5′-phosphate; UDP, uridine-5′-diphosphate; NAD , nicotinamide ade-
2
1
nine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; Glc, glucose; P -methyl-UDP, P -5′-
uridine-P -methyl diphosphate; Mops, 3-[N-morpholino]propanesulfonic acid; Tris, tris[hydroxymethyl]
2
aminomethane; DEAE, diethylaminoethyl; NMR, nuclear magnetic resonance.
2
9
0
045-2068/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

3
0
LIU, ARABSHAHI, AND FREY
+
+
dence: (1) The NAD in epimerase-NAD is rapidly reduced by D-glucose, D-fucose,
L-arabinose, D-xylose, D-galactose, or NaBH CN in the presence but not in the
3
+
absence of UMP (3–5). (2) Epimerase-NAD can also be reduced by NaBH in the
4
absence of UMP; however, the resulting epimerase-NADH undergoes autoxidation in
the air (6). (3) UMP both stabilizes epimerase-NADH against oxidation and stimulates
+
31
the reduction of epimerase-NAD by NaBH CN (6). (4) Electronic absorption and P
3
NMR spectroscopic results suggest that the conformation of epimerase is altered upon
uridine nucleotide binding (7,8). (5) The uridine pyrophosphoryl moiety of UDP and
2
13
15
P -methyl-UDP perturbs the C- and N-NMR chemical shifts of C4 and N1 in the
+
nicotinamide ring of epimerase-NAD . These perturbations correspond to a positive
change in the reduction potential and increased kinetic reactivity toward reducing
agents, as shown by studies of model compounds (2).
The objective of the present research was to quantify the kinetic activation of NAD
brought about by uridine nucleotides binding to the active site of epimerase-NAD and
+
+
13
to compare the observed effects with those predicted by the correlation of C NMR
perturbations with chemical reactivity in model, nonenzymatic reactions (2,9).
EXPERIMENTAL PROCEDURE
Glucose, UMP, UDP, and NaBH CN were purchased from Sigma. Glucose was
3
used as supplied, and UMP and UDP were purified by chromatography over DEAE-
Sephadex. Sodium cyanoborohydride was recrystallized, and the concentrations of stock
solutions were determined by titration with iodine (10). UDP-galactose 4-epimerase for
kinetic measurements was subjected to the denaturation-renaturation procedure to re-
move abortive complexes, as described elsewhere (7).
+
Rates of UMP- or UDP-dependent reduction of epimerase-NAD by glucose were
measured under aerobic conditions. Pseudo-first-order rate constants were determined
by fitting the time course of increasing A344 to the first-order rate equation. Rates were
measured in 25 mM K-phosphate buffer at pH 7.0 and 27°C at an ionic strength of 0.2
adjusted with KCl. In UMP-dependent reduction by glucose, the concentration ranges
were 0.1 to 1 M for glucose and 0.108 to 3.57 mM for UMP. In UDP-dependent reduc-
tion by glucose, the concentration ranges were 0.13 to 0.5 M for glucose and 0.03 to
0
.13 mM for UMP. The pseudo-first-order rate constants for reduction in the presence
of UMP and UDP were fitted to Eq. [1].
k[A][B]
kobs
=
[1]
[
A][B] + K [B] + K [A]
A
B
+
Uridine nucleotide-independent reduction of epimerase-NAD by glucose was car-
ried out anaerobically. Reaction solutions minus epimerase were made anaerobic by
purging with argon for 1 h, and the enzyme solutions were stirred under argon for 1.5
h. Reduction reactions were carried out in anaerobic cuvettes, in which the oxygen-
free solutions were mixed. The initial rates were measured spectrophotometrically at
3
44 nm at 27°C, either at pH 7.0 in 50 mM Mops buffer or at pH 8.5 in 0.1 M Tris
buffer, with the ionic strength set at 0.2 by addition of KCl. After sufficient time to
determine the initial rate of A -increase, UMP was added to the solutions to promote
344

+
REDUCTION OF NAD BY UDP-GALACTOSE 4-EPIMERASE
31
the complete reduction to epimerase-NADH-UMP. The total change in A344 after addi-
+
tion of UMP was used to calculate the initial concentration of epimerase-NAD , using
−1
−1
the published extinction coefficient of 6.22 mM cm (7). The initial rates were cal-
culated from the concentrations of enzyme and the initial ∆A344 at glucose concentra-
tion from 0.3 to 2.5 M. The data were plotted as initial rate divided by the concentra-
+
tion of epimerase-NAD against the concentration of glucose (Fig. 1).
FIG. 1. Uridine nucleotide-independent reduction of epimerase-NAD+ by glucose. Initial rates
acquired under anaerobic conditions at various glucose concentrations are plotted against glucose concen-
trations. Rates were measured spectrophotometrically at 344 nm. After measurement of the initial rate of
+
A
344-increase, UMP was added to stimulate the complete reduction of epimerase-NAD , and the total
increase in A344 was used to calculate the total concentration of enzyme in that experiment. The results
were then plotted as v /[E ] against [glucose]. The curves were obtained by fitting the data to Eq. [5]. (A)
Reduction carried out in 50 mM Mops, ionic strength 0.2, pH 7.0, at 27°C. (B) Reduction carried out in
.1 M Tris, ionic strength 0.2, pH 8.5, at 27°C.
i
o
0

3
2
LIU, ARABSHAHI, AND FREY
+
Reduction of epimerase-NAD by NaBH CN was carried out in a buffer consisting of
3
Mes, Mops, and N-methylpiperidine at 25, 25, and 50 mM, respectively, at pH 7.0 and
7°C. The ionic strength was adjusted to 0.2 by addition of KCl. UMP-independent
2
reduction was carried out in an anaerobic cuvette, with solutions deoxygenated as de-
scribed above. All kinetic data were acquired by use of a Hewlett-Packard Model 8452
spectrophotometer.
RESULTS
+
Uridine nucleotide-dependent reduction of epimerase-NAD by glucose. Pseudo-
+
first-order rate constants for UMP- or UDP-dependent reduction of epimerase-NAD
by glucose at pH 7.0 were first measured at glucose concentrations in the range of 0.1
to 1.5 M. It had been shown that at higher glucose concentrations the kinetics became
complex at pH 8.5. We confirmed the earlier reported rates at pH 8.5 (11), in which
−1
−1
downward curvature in plots of kobs against [Glc] was observed whenever glucose
concentrations exceeded 1.6 M. The earlier studies had been, therefore, confined to
less than 1.6 M glucose, where the double reciprocal plots were essentially straight
lines (11). Therefore, glucose concentrations from 0.1 to 1.0 M were chosen for uri-
dine nucleotide-dependent reductions at pH 7.0.
The dependence of the observed first-order rate constants on concentrations of glu-
cose and UMP or UDP was consistent with ordered binding of the uridine nucleotide
+
followed by glucose and then reduction of NAD by glucose to form the epimerase-
NADH-nucleotide complex according to Eqs. [2–4].
K
nuc
 
+
+

 
E-NAD + nucleotide
E-NAD -nucleotide
[2]
[3]
←
K
←
Glc
+
+

 
E-NAD -nucleotide + Glc
E-NAD -nucleotide-Glc
 
k
+
E-NAD -nucleotide-Glc  E-NADH-nucleotide + gluconolactone [4]
Based on earlier reports, the uridine nucleotide remains bound to the reduced enzyme
under the conditions of our experiments (12,13), and the glucose is oxidized at the
reducing carbon to form gluconolactone (11). UMP has been extensively studied as
+
the activator for reduction of epimerase-NAD by sugars; however, we have found
that UDP also potentiates the reaction, albeit at a slower rate. The kinetic parameters
in Table 1 were obtained for the UMP- and UDP-dependent reductive inactivation of
TABLE 1
Kinetic Parameters for Uridine Nucleotide-Dependent
+
Reduction of Epimerase-NAD by Glucose
Parameter
UMP, pH 7.0
UDP, pH 7.0
−
1
k (min )
K
K
15.6 ± 1.2
0.88 ± 0.03
2.9 ± 0.3
1.24 ± 0.11
0.084 ± 0.001
2.9 ± 0.3
nucleotide (mM)
Glc (M)

+
REDUCTION OF NAD BY UDP-GALACTOSE 4-EPIMERASE
33
epimerase. The maximum rate for UMP-dependent reduction at pH 7.0 is somewhat
faster than in the earlier study at pH 8.5 (11).
Uridine nucleotide-independent reduction by glucose. In order to measure the mag-
+
nitude of the reductive activation of NAD brought about by binding uridine nucleotides
+
to epimerase-NAD , it is necessary to measure the rate in the absence of a uridine
+
nucleotide. Reduction of epimerase-NAD by glucose alone has not been observed in
the past. Because epimerase-NADH produced by reduction with NaBH in the ab-
4
sence of a uridine nucleotide undergoes spontaneous reoxidation in air, and this does
not occur under anaerobic conditions (6), it is possible that reduction by glucose at a
very slow rate would not be observable in the air because of spontaneous reoxidation
of epimerase-NADH. Therefore, we undertook to determine whether glucose would
+
reduce epimerase-NAD in the absence of dioxygen.
We have been able to observe the reduction of epimerase-NAD by, D-glucose at a
+
slow rate by monitoring the increase in A344 under anaerobic conditions. The reason
that reduction by glucose was not observed in the past appears to be that reduction is
slower than reoxidation in the air, and reduction can be observed only under anaero-
bic conditions. When a uridine nucleotide is present, the reduced enzyme is protected
from reoxidation by air, so that uridine nucleotides both potentiate the reduction and
protect the reduced enzyme from reoxidation (6).
+
Because the uridine nucleotide-independent reduction of epimerase-NAD is very
slow, it is impractical to observe the complete reduction to epimerase-NADH, a process
that would require several days. Therefore, we measured the initial rates of reduction as
a function of glucose concentration at pH 7.0. Shown in Fig. 1 are the data obtained for
+
the reduction of epimerase-NAD by glucose at pH 7.0 and pH 8.5. The data are plotted
as initial rates divided by epimerase concentration against glucose concentrations. The
data neither show a direct proportionality, which would be observed in ordinary first-
order dependence on glucose concentrations, nor a leveling off at increasing concentra-
tions that would be observed in saturation kinetics. Instead, an upward curvature is
observed at both pHs with increasing glucose concentrations.
Owing to the complexity of the graphs in Fig. 1, the data were fitted to Eq. [5], in
which
2
v /[E ] = k [Glc] + k [Glc]
[5]
i
o
1
2
+
[
E ] is the initial concentration of epimerase-NAD , v is the initial rate of its reduc-
o
i
tion to epimerase-NADH, and k is the second order rate constant for the reduction of
1
+
epimerase-NAD by glucose. Equation [5] represents the rate law for reduction in the
absence of uridine nucleotides, and the data indicate that a second molecule of glucose
is involved in the reaction at high glucose concentrations. We found that the data were
2
not adequately described by the equation v /[E ] = k [Glc] , that is by a strict second-
i
o
1
order dependence on glucose concentration. Therefore, the first term in Eq. [5] is an
essential part of the rate law, and the second term increases the reaction rate but is not
essential for the reaction.
The squared term may be mechanistically rationalized in any of several ways. For
example, it may be that a second molecule of glucose can bind to the uridine nucleotide
subsite and activate the reduction in the same way as UMP or UDP. Alternatively, both

3
4
LIU, ARABSHAHI, AND FREY
glucose molecules may bind in the glycosyl subsite and potentiate the reaction. How-
ever a second molecule of glucose may function, the objective of the present study is
limited to the evaluation of k governing the reaction of a single molecule of glucose
1
+
with epimerase-NAD .
The first coefficient in the rate law represents the second order rate constant for the
+
bimolecular reduction of epimerase-NAD by glucose. The values of k for reduction at
1
−4
−1
−1
2
7°C, resulting from the fits to the rate law in Fig. 1, are (3.9 ± 1.2) × 10 M min at
pH 7.0 and (1.3 ± 0.2) × 10 M min at pH 8.5.
−3
−1
−1
+
Reduction of epimerase-NAD by NaBH CN. Sodium cyanoborohydride is much
3
less reactive than sodium borohydride and, as in reduction by glucose, reduction of
+
epimerase-NAD by cyanoborohydride is not observed under aerobic conditions. It is,
however, observed under anaerobic conditions. The second-order rate constant for
+
reduction of epimerase-NAD by NaBH CN was by the same procedure employed for
3
−1
−1
the reaction of glucose and found to be 0.22 ± 0.04 M min . In the case of
NaBH CN, the initial rates were directly proportional to the concentration of reduc-
3
tant. In the presence of 5 mM UMP (88% saturation), the second-order rate constant
−1
−1
−1
−1
was 166 ± 9 M min , which corresponded to 190 M min at saturation with UMP.
Therefore, the rate enhancement brought about by UMP was 860-fold.
DISCUSSION
A kinetic mechanism for uridine nucleotide-independent reduction of epimerase-
+
NAD . In the earlier kinetic analysis of UMP-dependent reduction by glucose at
pH 8.5, a random equilibrium binding mechanism was found for UMP and glucose (11).
However, the binding of glucose and UMP were significantly synergistic, such that in
the preferred binding pathway UMP was bound first followed by glucose. Glucose
was very weakly bound with a K of 3 M for dissociation from the enzyme and 0.5 M
d
from the epimerase–UMP complex. Selected data from the earlier work at pH 8.5
were replicated in this study and found to be accurate. In the present work at pH 7.0,
the data showed that binding of glucose was more synergistic than at pH 8.5, such that
both UMP- and UDP-dependent reduction by glucose appeared to follow essentially
ordered binding mechanisms, and the data were fitted to the rate equation for ordered
binding, with the nucleotide leading.
A kinetic mechanism for the UMP-independent reduction by glucose must be con-
sistent with Eq. [5], to which the data in Fig. 1 have been fitted. A mechanism in which
+
the reduction of epimerase-NAD is preceded by the binding of glucose is described
+
by Eqs. [6] and [7], in which E = epimerase-NAD and E-H = epimerase-NADH. The
mechanism includes two glucose binding
K
g
1
1
k

 
E-Glc    E-H + gluconolactone
E + Glc
[6]
[7]
←
 
K
g
2
2
k

 
E-Glc  E-H + gluconolactone
E-Glc + Glc
←
 
2
interactions and two rate constants kg1 and kg2 for reduction. We are interested in the first
rate constant for comparison with the value of k in the uridine nucleotide-dependent reduc-
tive inactivation. The rate law pertaining to the mechanism of Eqs. [6] and [7] is Eq. [8],
1 2
where K and K

+
REDUCTION OF NAD BY UDP-GALACTOSE 4-EPIMERASE
35
vi
kg1
kg2
=
[Glc] +
[Glc]2
[8]
[
Eo ] K1
K2
1 2 1 2
are dissociation constants for glucose. In Eq. [8], kg1 /K and kg2 /K correspond to k and k ,
respectively, in Eq. [5].
In the mechanism of Eqs. [6] and [7], kg /K for uridine nucleotide-independent reduc-
1
1
tion is the second-order rate constant for reduction of epimerase by glucose. Inasmuch
as saturation by glucose was not observed in the initial rate measurements, it might be
appropriate to treat the data as if glucose binding did not take place prior to reduction of
the enzyme. However, in the UMP-dependent reduction at pH 8.5, binding of glucose to
the free enzyme was clearly observed (11). At pH 7.0 an ordered equilibrium binding
mechanism with the uridine nucleotide binding first dominates the kinetics, owing to the
higher affinity of the enzyme for uridine nucleotides at pH 7.0 compared with pH 8.5.
However, we believe that glucose binds to the free enzyme at pH 7.0, but the binding of
glucose does not appear in the kinetics because of being on a minor pathway. In the
absence of a uridine nucleotide in the experiments of Fig. 1, weak binding of glucose
would be obscured by the squared term in the rate law. In any case, kg /K is the second-
1
1
order rate constant for uridine nucleotide reduction by glucose, and it can be compared
with k/KGlc for uridine nucleotide-dependent reduction.
Magnitude of rate enhancement by uridine nucleotides. The rate enhancement for
glucose-reduction brought about by a uridine nucleotide is given by the ratio of the
second-order rate constant for the reaction of glucose with the enzyme-uridine
nucleotide complex to the second-order rate constant for the reaction of glucose with the
free enzyme. The ratios k/KGlc calculated from the parameters in Table 1 give the sec-
ond-order rate constants for uridine nucleotide-dependent reduction, and the values for
nucleotide-independent reduction (kg
1
/K ) quoted under Results are derived from the fits
1
in Fig. 1. The ratio of these rate constants for UMP-dependent reduction at pH 7.0 is
−1
1
4,000, which corresponds to a decrease in activation energy of 5.7 kcal mol that is
brought about by UMP. In the case of UDP, the ratio of rate constants is 1100, which
−1
corresponds to a decrease in activation energy of 4.1 kcal mol . Kang et al. (11) found
−1
the maximum rate constant and KGlc to be 6.3 min and 0.5 M, respectively, at pH 8.5,
−1
−1
corresponding to a second-order rate constant of 13 M min . The second-order rate
−
3
constant for UMP-independent reduction at pH 8.5 in the present work is 1.3 × 10
−1
−1
M min . Therefore, UMP enhances the rate 10,000-fold at pH 8.5, which corresponds
−1
to a decrease in the activation energy of 5.5 kcal mol .
The rate enhancements calculated here include both the effects of glucose binding and
the conformational change brought about by UMP (or UDP). It seems that the major part
of the rate enhancement can be allotted to the effects of the conformational change on
+
the reactivity of NAD at the active site. This interpretation is supported by the fact
that UMP stimulates the rate at which the nonspecific reductant NaBH CN reduces
3
+
epimerase-NAD by 860-fold. Therefore, UMP decreases the activation energy by
−1
−1
4
.0 kcal mol in the reduction by NaBH CN at pH 7.0, about 1.5 kcal mol less than
3
the reduction by glucose, so that more than 70% of the enhancement can be allotted
+
to the effects of the conformational change on the intrinsic reactivity of NAD .
Correlations of uridine nucleotide-induced rate enhancements with polarization of
+
the electronic structure in NAD . Uridine nucleotide-dependent reductive inactivation

3
6
LIU, ARABSHAHI, AND FREY
of UDP-galactose 4-epimerase is attributed to an induced conformational change in
+
the enzyme, which increases the chemical reactivity of NAD at the active site. The
13
15
conformational change induces perturbations in the C- and N-NMR chemical shifts
+
of atoms in the nicotinamide ring of epimerase-NAD (2). An upfield perturbation of
1
5
15
+
3
.0 ppm for the N-NMR signal of epimerase-[1- N] NAD and a 3.4 ppm downfield
1
3
13
+
perturbation for the C-NMR signal of epimerase-[4- C] NAD were induced by
binding UDP (2). To evaluate the kinetic significance of these perturbations, the chem-
ical reactivities and NMR properties of a series of N-alkyl nicotinamides with a range
of inductive electron withdrawing N-alkyl substituents were studied (2,9). The chem-
ical shifts were correlated with the reduction rates by NaBH CN and also the reduc-
3
tion potentials, and the results were used to evaluate the probable kinetic significance
13
of uridine nucleotide-induced perturbations in the [4- C]nicotinamide chemical shifts
1
3
+
of epimerase-[4- C] NAD . The kinetic correlations showed that a 3.4-ppm downfield
1
3
13
perturbation of the C NMR signal for an N-alkyl-[4- C]nicotinamide corresponded
to a 3,000 to 15,000-fold enhancement in the rate of reduction by NaBH CN, depend-
3
13
ing on the polarity of the solvent. The 3.4-ppm perturbation in the C-NMR chemical
shift also corresponded to a 150 mV more positive reduction potential for an N-alkyl-
nicotinamide.
Until now, the magnitude of rate enhancement brought about by UMP and UDP in
+
the reduction of epimerase-NAD was not known. Because reduction by sugars in the
absence of a uridine nucleotide could not be observed until now, the rate enhancement
10
could in principle have been any value within the range of 10 to 10 or higher. The
+
earlier correlations could account for the purely chemical activation of NAD , but they
+
did not prove that the UDP-induced increase in the chemical reactivity of NAD asso-
+
ciated with epimerase-NAD accounted for all or most of the rate enhancement.
The rate increases of 1,100- and 14,000-fold induced by UDP and UMP are very
13
well correlated with the C NMR results on epimerase and with the NMR and kinetic
results on N-alkylnicotinamides (2). Thus, the uridine nucleotide-induced polarization
+
of the π-electrons in the nicotinamide ring of epimerase-NAD , as observed by NMR,
accurately report on the conformationally induced rate enhancement.
The nature of the conformational change leading to the polarization of the π-electrons
+
in NAD is not known. The available X-ray structures of UDP-galactose 4-epimerase
include a uridine nucleotide in the active site (14–16). Kinetic evidence implicates Lys
1
53 and Tyr 149 in this phenomenon (17,18). Lys 153 binds the 2′- and 3′-hydroxyl
+
groups of the nicotinamide riboside portion of NAD , and Tyr 149 engages in charge
transfer complexation with the nicotinamide ring of NAD (7,18). A complete descrip-
+
tion of the nature of the conformational change and the physicochemical basis for the
polarization of the nicotinamide ring await the structure of the free enzyme.2
ACKNOWLEDGMENT
This research was supported by Grant GM 30480 from the National Institute of General Medical
Sciences, USPHS.
2
+
A referee suggested that the uridine nucleotide might interact with the carboxamide group of NAD in
the active site and thereby regulate reactivity at C4. We regard this as unlikely for two reasons. All X-ray
structures show that the uridine diphosphate group does not interact with the carboxamide group or any
+
15
15
+
other part of NAD (14–16,18). N NMR spectroscopy of epimerase-[carboxamide- N]NAD shows that
the binding of UDP does not perturb the carboxamide group (2).

+
REDUCTION OF NAD BY UDP-GALACTOSE 4-EPIMERASE
37
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1
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(
2
3
4
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6
7
8
. Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220–12230.
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