Catalytic mechanism of human UDP-glucose 6-dehydrogenase: In situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction

Article abstract of DOI:10.1016/j.carres.2012.03.028

Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD⁺-dependent steps via covalent thiohemiacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through attack of Cys²⁷⁶ on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu¹⁶¹→Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (≤2%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by thiohemiacetal adduct formation.

Full text of DOI:10.1016/j.carres.2012.03.028

Carbohydrate Research 356 (2012) 209–214
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Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ
proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not
exchanged with bulk water during the enzymatic reaction
Thomas Eixelsberger a, Lothar Brecker b, Bernd Nidetzky a,
⇑
a Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria
b Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into
UDP-glucuronic acid. The catalytic reaction proceeds in two NAD+-dependent steps via covalent thiohem-
iacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through
attack of Cys276 on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because pre-
vious studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-
gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into
product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting
Glu161?Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measure-
ments showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transforma-
tions. The product contained no deuterium at C-5 within the detection limit (62%). The results are
consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is
immediately trapped by thiohemiacetal adduct formation.
Received 1 February 2012
Received in revised form 23 March 2012
Accepted 24 March 2012
Available online 2 April 2012
Keywords:
UDP-glucose dehydrogenase
Thiohemiacetal and thioester intermediates
Catalytic mechanism
Deuterium incorporation
UDP-gluco-hexodialdose
Ó 2012 Elsevier Ltd. All rights reserved.
UDP-glucuronic acid (UDP-GlcUA) is an important metabolite of
cellular physiology. It also serves a role in tissue development, be-
cause biosynthesis of extracellular matrix glycosaminoglycans in
vertebrates requires UDP-GlcUA as precursor.1 UDP-GlcUA is de-
rived from UDP-glucose (UDP-Glc) by two-step NAD+-dependent
oxidation of the C-6 alcohol, catalyzed by UDP-glucose 6-dehydro-
genase (UGDH).2 Genes encoding UGDH are present throughout all
domains of life. Analysis of UGDH amino acid sequences reveals
that enzymes from different lineages clearly share common ances-
try.2 Basic elements of structure and function are therefore con-
served among the different UGDHs.3,4 All known UGDHs fold into
oligomers.2 The homodimeric quaternary structure adopted by
bacterial enzymes appears to be the basic unit of UGDH enzyme
function.3,5,6 Enzymes from eukaryotic origin form more complex
ring-shaped homohexameric assemblies.4,7–10 The role of the par-
ticular structural architecture of UGDH from higher organisms is
not entirely clear, but it could confer certain features of allosteric
regulation.7,8 Residues of the active site are highly conserved
among the different UGDHs, with only few exceptions.2 The cata-
lytic mechanism of UGDH therefore seems to be highly conserved.
Enzymatic oxidation of UDP-Glc occurs in three catalytic steps via
covalent thiohemiacetal and thioester intermediates, as shown in
Scheme 1 and Fig. 1.3,4,10–16 The proposed basic mechanism of
UGDH is well supported by evidence from crystal structures and
biochemical data for the wild-type enzyme and relevant mutants
thereof.
Mechanistic proposals for UGDH differ in an important detail
that is the kinetic significance of the enzyme-bound product of
the first oxidation step, namely UDP-gluco-hexodialdose.12,13 Stud-
ies of UGDH from different sources concur that not even traces of
UDP-gluco-hexodialdose are released from the enzyme during the
reaction. One possibility considered was that the incipient UDP-
gluco-hexodialdose is immediately trapped at the active site as a
covalent thiohemiacetal adduct (Scheme 1).4 Hydride transfer oxi-
dation by NAD+ and nucleophilic addition of the thiolate side chain
of cysteine to the reactive C-6 of substrate would therefore take
place in a kinetically coupled fashion, effectively reducing the life-
time of any UDP-gluco-hexodialdose formed in the reaction. We
refer to this mechanism as concerted throughout the paper. The
mechanistic idea is supported by kinetic data for human UGDH
(hUGDH) from this laboratory, demonstrating that enzyme depro-
tonation, presumably at the active-site cysteine (Cys276), occurs
kinetically in parallel with the initial substrate oxidation.4 More-
over, total disruption of covalent catalysis by Cys276 in a Cys?Ala
mutant resulted in the massive impairment of already the first oxi-
dation step.4,10 If the cysteine was required only in the second oxi-
dation step, one would expect that a Cys?Ala mutant accumulates
⇑
Corresponding author. Tel.: +43 316 873 8400; fax: +43 316 873 8434.
E-mail address: bernd.nidetzky@tugraz.at (B. Nidetzky).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.03.028

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T. Eixelsberger et al. / Carbohydrate Research 356 (2012) 209–214
Scheme 1. Proposed course of the catalytic reaction of UGDH. For clarity reasons, only the herein relevant amino acids of the active site of hUGDH are shown. The concerted
mechanism would be inconsistent with exchange of the proton at glucosyl C-5 with solvent. The stepwise mechanism might allow proton exchange due to accumulation of
enzyme-bound UDP-gluco-hexodialdose. Unless UDP-gluco-hexodialdose was released from the active site (which probably does not happen as shown later), exchange
reaction would have to take place at the active site of the enzyme.
steps (stepwise mechanism). Release of UDP-gluco-hexodialdose
from the enzyme complex with NADH may not be possible due to
the (closed) protein conformation adopted at this point of the reac-
tion.3,4 In support of this mechanistic view, chemically synthesized
UDP-gluco-hexodialdose was a fairly good substrate for oxidation
(with NAD+) or reduction (with NADH) by UGDH.12,13,15,18 Working
with UGDH from the bacterium Streptococcus pyogenes, Tanner and
co-workers showed that the wild-type enzyme, but also a mutated
variant that had the key cysteine (Cys260) replaced by Ala, catalyzed
oxidation and reduction of UDP-gluco-hexodialdose.12 These
authors therefore concluded that the cysteine had no catalytic role
in the first oxidation step.12 They also proposed that NADH was re-
leased from the enzyme complex with UDP-gluco-hexodialdose
only after formation of the covalent thiohemiacetal adduct (Scheme
1). In their mechanistic studies of bovine liver UGDH, Feingold and
colleagues observed that tritium label from C-5 of UDP-Glc was
partly ‘washed out’ to solvent during the enzymatic reaction.19 Loss
of label (30% of the original tritium) was explained by an enoliza-
tion of the C-6 aldehyde in UDP-gluco-hexodialdose prior to cova-
lent adduct formation (Scheme 2). No exchange of tritium was
thought to occur in the thiohemiacetal and thioester intermediates.
The proposed keto-enol tautomerism at the UGDH active site re-
quires the C-6 aldehyde to become a rather long-lived intermediate
of the enzymatic reaction and therefore, it would be clearly
inconsistent with the idea of a strong kinetic coupling between
substrate oxidation and thiohemiacetal formation. Utilization of
UDP-gluco-hexodialdose as substrate for oxidation and reduction,
by contrast, could be reconciled with the concerted mechanism,
because the observed activities may not reflect exactly the steps
that occur in the normal reaction pathway. Using UDP-galacto-
hexodialdose (prepared from UDP-galactose by oxidation with
galactose oxidase) as surrogate of UDP-gluco-hexodialdose, the
authors demonstrated that the C-5 tritium readily exchanges with
the solvent proton in a spontaneous (non-enzymatic) reaction.19
In contrast, chemically synthesized UDP-gluco-hexodialdose forms
Figure 1. Close-up representation of the active site of E161Q in
a trapped
thiohemiacetal enzyme intermediate. Cys276 is the site of covalent modification.
The PDB code of the structure is 3KHU.
a detectable amount of NADH in a pre-steady burst, which results
due to the relatively fast conversion of UDP-Glc to UDP-gluco-
hexodialdose. Even though alcohol oxidation by NAD+ proceeds
thermodynamically uphill, the non-favorable position of the reac-
tion equilibrium would not prevent the formation of the aldehyde
product as long as NAD+ and UDP-Glc are present in excess. Note
that aldehyde reductases readily oxidize primary alcohols by
NAD+ or NADP+.17 It is also relevant that trapping of the UDP-glu-
co-hexodialdose by chemical reduction with NaBH4 proved fruit-
less unless the active-site cysteine (of bovine liver UGDH) had
been inactivated by chemical modification with cyanide.14
The alternative possibility is that substrate aldehyde and cova-
lent thiohemiacetal adduct are formed in two sequential kinetic

T. Eixelsberger et al. / Carbohydrate Research 356 (2012) 209–214
211
Scheme 2. Enolization and proton exchange at C-5 of UDP-gluco-hexodialdose. If the aldehyde enolized prior to covalent intermediate formation, tritium label at C-5 could be
replaced by hydrogen through label ‘wash-out’ to solvent. Similarly, deuterium could be incorporated (‘washed in’) if the reaction was performed in D2O.
a hydrate upon contact with water, as shown by Campbell and
Tanner.18 No enolization or C-5 proton exchange was observed in
their study.
low overall activity of the mutant in the experiment. Substrate
conversion did not exceed 20% after extended incubation times.
However, it was sufficient to characterize the stoichiometry of
the reaction and to determine the relevant proton signals in the
UDP-GlcUA product. No UDP-gluco-hexodialdose was detectable
in the reaction of E161Q.
Considering the mechanistic significance of the problem, and
the partially contradictive results published so far, we decided to
re-examine in this work the reported hydrogen exchange at C-5
during enzymatic oxidation of UDP-Glc. We took an approach dis-
tinct from that of Feingold and co-workers19 in that we measured
incorporation of solvent deuterium by the UDP-GlcUA product. In
situ proton NMR spectroscopy was employed to observe the enzy-
matic transformations in real time. We performed experiments
with two forms of hUGDH, the wild-type enzyme and a point mu-
tant that had Glu161 replaced by Gln (E161Q). Reason to compare
the two enzymes was that each represented a distinct rate-deter-
mining step.4,10 Glu161 is the general catalytic base for hydrolysis
of the thioester intermediate in the proposed mechanism of hUG-
DH (Scheme 1). The E161Q mutant is particularly impaired in the
hydrolysis step and therefore accumulates the thioester adduct at
steady state. The mutant is about 600 times less reactive in terms
of turnover number (kcat) than wild-type hUGDH.10 In the wild-
type enzyme at non-saturating concentration of NAD+ (0.5 mM),
the thiohemiacetal intermediate accumulates at a steady state be-
cause the overall exchange of NADH by NAD+ becomes rate limit-
ing.4 At saturating concentration of NAD+, none of the different
catalytic steps in Scheme 1 appears to be distinctly rate determin-
ing in wild-type hUGDH. We considered that if hydrogen/deute-
rium exchange at C-5 of enzyme-bound UDP-gluco-hexodialdose
took place at all in hUGDH, we should be able to accumulate suffi-
cient amounts of the relevant intermediate in one of the two
enzymes to make the exchange reaction eventually observable in
the experiment. We performed in situ NMR experiments at pD
7.5 (wild type) and 8.3 (E161Q), because hUGDH is active and sta-
ble under these conditions. The previous work with bovine liver
UGDH was done at a higher pH of 8.7.19 We therefore analyzed
deuterium incorporation into the UDP-GlcUA product at different
pD values in the range 5.9–8.8, using NMR measurement at a single
time point of the reaction.
The 1H NMR signal of proton H-4 in resulting UDP-GlcUA ap-
pears at 3.50 ppm. Although it is slightly overlapped (Fig. 2A), its
3
doublet of doublet structure is well detectable and the JH-H cou-
pling constants can be determined to be 10.2 Hz and 9.2 Hz,
respectively. Such quite large couplings indicate H-4 as well as
both neighbored protons H-3 and H-5 to be in axial positions.
The H-5 is hence not exchanged by deuterium. Furthermore, no
3
3
additional signal of H-4 with one JH-H to H-3 and a JH-D to H-5
can be detected. The concomitant slight isotope shift would cause
such signal to be in the spectral region of 0.015 ppm around that
of H-4 in the fully protonated UDP-GlcUA. This region is not over-
lapped by signals of further protons and hence allows a detection
even of small amounts of byproducts. The absence of such signal
can hence be used to exclude possible H/D exchange in an extent
of more than 1%, which is the detection limit.
We analyzed the development of signal intensity of selected
protons from UDP-GlcUA (H-1, H-4, H-5) in dependence of the
incubation time. The results are summarized in Figure 3 for the
reaction of wild-type UGDH (panel B) and E161Q (panel D). There
was no loss of H-5 signal within error limit of 2%, which also indi-
cates that exchange with the solvent had not occurred. Addition-
ally, we tested the effect of lowering the concentration of NAD+
from 15 mM, which is completely saturating, to just 0.5 mM, which
is limiting at the steady state, on H-5 signal evolution in product. It
was shown in recent work that at 0.5 mM NAD+ the overall oxida-
tion of thiohemiacetal intermediate becomes rate determining.4
We considered that under the conditions of limiting NAD+ the pro-
ton exchange reaction might therefore be favored. The reaction
was performed for that reason directly in the NMR tube and
NAD+ was supplemented in regular intervals to promote the con-
version. NMR data showed that H-5 signal intensity was not af-
fected as compared to reference proton signals.
Figure 2 shows time-resolved proton NMR data recorded for
oxidation of UDP-Glc by wild-type hUGDH (panel A) and E161Q
(panel B). Results are presented in stack plots of seven selected
spectra acquired over a reaction time of 12 h. Some selected signals
from the UDP-Glc, UDP-GlcUA and NADH were assigned according
to the literature and allow unambiguous identification of the reac-
tion products. No intermediate UDP-gluco-hexodialdose was ob-
served within detection limit, as expected. The presumed
stoichiometry of the enzymatic transformation that two NADH
and one UDP-GlcUA are produced for each UDP-Glc oxidized was
confirmed in a quantitative analysis of the NMR data, as shown
in Figure 3 (panels A and C). The rather low conversion of UDP-
Glc observed in our experiments can be explained by a large sol-
vent isotope effect on kcat for the respective enzyme used, resulting
in a fourfold decreased reaction rate in D2O as compared to the cor-
responding reaction rate in water under the conditions applied.
This large solvent isotope effect could deserve further attention
in future mechanistic studies of hUGDH. The intrinsical low activ-
ity of E161Q combines with the isotope effect on kcat to give a very
We also examined the effect of pH on the protonation state of C-
5 in UDP-GlcUA. Reactions were performed in D2O at pD 5.9, 7.0,
7.9, and 8.8. Conversion of UDP-Glc substrate increased in response
to a raise of pD, as one might expect from the known pH dependen-
cies of kcat for wild-type enzyme and E161Q (Fig. 4). Both enzymes
show optimum activity at pH 8 or higher.4,10 Under each of the
conditions used, neither enzymatic transformation went along
with a relative decrease in H-5 signal. We therefore conclude that
incorporation of solvent deuterium at C-5 did not take place in the
course of reactions catalyzed by wild-type enzyme and E161Q.
In summary, evidence from this work does not support the
mechanistic proposal for UGDH (Scheme 2) that the UDP-gluco-
hexodialdose intermediate of the enzymatic reaction partly
exchanges its proton at C-5 with bulk solvent due to keto-enol
tautomerism. The original observation made with bovine liver
UGDH that tritium label at C-5 of UDP-Glc was partly washed
out during reaction19 was re-examined using an alternative,
equally diagnostic approach in which deuterium uptake from

212
T. Eixelsberger et al. / Carbohydrate Research 356 (2012) 209–214
Figure 2. In situ NMR spectroscopic measurement of hUGDH catalyzed oxidation of UDP-Glc to UDP-GlcUA. The data are presented as stack plot of seven selected spectra in
regular intervals of 2 h. Indicative signals of NADH, UDP-Glc, and UDP-Glc-UA are marked and assigned according to literature.24 Signals of impurities (TRIS from enzyme
preparations, ethanol from commercial NAD+) are indicated with asterisks. No signals indicating presence of UDP-gluco-hexodialdose could be detected. Panels A and B show
the reaction of wild-type hUGDH and E161Q, respectively. The spectral region from 3.89 ppm to 3.52 ppm is not shown in panel B due to presence of larger signals from
impurities (glycerol). In panel A, the area of the spectrum showing the 1H NMR signal of proton H-4 in UDP-GlcUA is also presented enlarged in a box.
solvent was measured. Instead of the bovine liver enzyme, the
structurally and mechanistically well-characterized hUGDH, both
in wild-type and Glu161?Gln mutated form, was used. There was
no deuterium incorporation, measured as relative H-5 signal loss,
under a range of conditions (pH, NAD+ concentration) that were
chosen to involve change in rate-determining step of the enzy-
matic reaction. Our observations are therefore consistent with
the idea of a concerted mechanism for the human enzyme (Scheme
1) that precludes proton exchange with solvent. They are however
also consistent with a sequential mechanism in which the interme-
diary UDP-gluco-hexodialdose is sequestered at the binding site of
the enzyme so that exchange with bulk solvent is prevented effec-
tively. We note however that protons are released from the active
site of hUGDH in the course of the first oxidation step, suggesting
that there exists probably connection between the catalytic center
and solvent during the reaction.4 We also note that chemically,
hydration of C-6 aldehyde might be a more likely outcome of
contact of UDP-gluco-hexodialdose with water than fast enoliza-
tion at around neutral pH. It is difficult to envision mechanistic dif-
ferences between hUGDH and the enzyme from bovine liver that
would reconcile the conflicting findings from deuterium incorpora-
tion and tritium ‘wash out’ experiments. Accumulation of UDP-glu-
co-hexodialdose as enzyme-bound intermediate in bovine liver
UGDH is a possibility. This would be not consistent however with
the proposed kinetic mechanism of the enzyme having thioester
hydrolysis as the rate determining step.20
1. Experimental
1.1. Materials
UDP-Glc (sodium salt) was purchased at Carbosynth. UDP-
GlcUA (ammonium salt; >98% purity), and NAD+ (sodium salt;
98% purity) were obtained from Sigma–Aldrich.

T. Eixelsberger et al. / Carbohydrate Research 356 (2012) 209–214
213
Figure 3. Time courses of enzymatic oxidations of UDP-Glc, and determination of the associated change of relative H-5 signal intensity in UDP-GlcUA product. Time courses
are shown in panels A (wild-type) and C (E161Q), the symbols used are square (UDP-Glc), triangle (UDP-GlcUA), and diamond (NADH). The low conversion can be explained
by a large solvent isotope effect on kcat (see text). Two equivalents of NADH are formed per equivalent UDP-Glc utilized, as expected. Proton signal intensities in UDP-GlcUA
are shown in panels B and D (square: H-1; triangle H-4; diamond: H-5) for reactions with wild-type enzyme and E161Q mutant, respectively. In case of deuterium
incorporation at C-5, one would see a decrease of the respective proton signal compared to H-1 and H-4.
1.2. Enzymes
Purified preparations of wild-type hUGDH and E161Q mutant
were obtained using reported procedures.4,10 Briefly, overexpres-
sion of the coding genes was done in Escherichia coli BL21(DE3)-
R3. Both enzymes were produced as fusion proteins containing an
N-terminal extension, which comprised a solubility enhancement
tag, a streptavidin tag, and a tobacco etch virus protease cleavage
site. Cell extracts were first purified by affinity chromatography.
The N-terminal extension was then removed, and the enzymes were
further purified by gel filtration and anion-exchange chromatogra-
phy. Full details of the purification protocol are given elsewhere.4,10
1.3. Assays
Enzymatic assays were performed in 1 mL of D2O. A 50 mM
potassium phosphate buffer at pD 5.9, 7.0, 7.9 or 8.8 was used.
The pD was determined as meter reading +0.4. Starting concentra-
tions were 15 mM NAD+, 2 mM UDP-Glc and 0.38
l
M wild-type
hUGDH or 15
lM E161Q. After addition of all components, the
Figure 4. pH dependence of conversion of UDP-Glc by wild-type hUGDH and
E161Q mutant. The symbols are filled circle (wild-type enzyme) and empty circle
(mutant). The reaction time was 16 h at 25 °C.
reactions were incubated at 25 °C for 16 h.
For determination of the solvent isotope effect, enzymatic con-
versions in H2O or D2O were performed in a Beckman Coulter

214
T. Eixelsberger et al. / Carbohydrate Research 356 (2012) 209–214
DU800 spectrophotometer using a 50 mM potassium phosphate
buffer with pH/pD 7.5. Reaction velocity was measured by NADH
absorption (k = 340 nm).
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1.5. Data analysis
The Topspin 3.0 software from Bruker was used for processing
the NMR spectra after data acquisition. For quantitative analysis
of the NMR data, a correction value was subtracted from the inte-
gral values of each proton to account for background noise.
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Financial support from the Austrian Science Fund FWF (Project
DK Molecular Enzymology W901-B05) is gratefully acknowledged.