Structural and kinetic evidence that catalytic reaction of human UPD-glucose 6-dehydrogenase involves covalent thiohemiacetal and thioester enzyme intermediates

Article abstract of DOI:10.1074/jbc.M111.313015

Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD⁺-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu¹⁶¹) by an incompetent analog (Gln¹⁶¹) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mM UDP-glucose and 2 mM NAD⁺, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 A resolution. Cys²⁷⁶ was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD⁺. The proposed catalytic mechanism of human UGDH involves Lys²²⁰ as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp ²⁸⁰ deprotonates Cys²⁷⁶ to function as an aldehyde trap and also provides oxyanion stabilization. Glu¹⁶¹ is the Bronsted base catalytically promoting the thioester hydrolysis.

Full text of DOI:10.1074/jbc.M111.313015

Enzymology:
Structural and Kinetic Evidence That
Catalytic Reaction of Human UDP-glucose
6-Dehydrogenase Involves Covalent
Thiohemiacetal and Thioester Enzyme
Intermediates
Sigrid Egger, Apirat Chaikuad, Mario
Klimacek, Kathryn L. Kavanagh, Udo
Oppermann and Bernd Nidetzky
J. Biol. Chem. 2012, 287:2119-2129.
doi: 10.1074/jbc.M111.313015 originally published online November 28, 2011
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 3, pp. 2119–2129, January 13, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Author’s Choice
Structural and Kinetic Evidence That Catalytic Reaction of
Human UDP-glucose 6-Dehydrogenase Involves Covalent
□
S
Thiohemiacetal and Thioester Enzyme Intermediates*
Received for publication, October 12, 2011, and in revised form, November 24, 2011 Published, JBC Papers in Press, November 28, 2011, DOI 10.1074/jbc.M111.313015
Sigrid Egger‡§, Apirat Chaikuad¶, Mario Klimacek‡, Kathryn L. Kavanagh¶, Udo Oppermann¶ʈ,
and Bernd Nidetzky‡§1
From the ‡Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, A-8010 Graz, Austria,
¶Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, United Kingdom,
ʈBotnar Research Centre, National Institute for Health Research Oxford Biomedical Research Unit, University of Oxford, Oxford
OX3 7LD, United Kingdom, and §Austrian Centre of Industrial Biotechnology, A-8010 Graz, Austria
Background: Human UDP-glucose 6-dehydrogenase (hUGDH) is responsible for biosynthesis of UDP-glucuronic acid.
Results: We report the crystal structure of a thiohemiacetal enzyme intermediate and demonstrate kinetic trapping of a
thioester enzyme intermediate.
Conclusion: Covalent catalysis by Cys276 is established. Glu161 functions as a Brønsted base for hydrolysis of the thioester
enzyme intermediate.
Significance: A detailed mechanism for covalent catalysis by hUGDH is proposed.
؉
Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-de- NAD . The proposed catalytic mechanism of human UGDH
hydrogenase (UGDH) occurs through the four-electron oxida- involves Lys220 as general base for UDP-glucose alcohol oxida-
؉
tion of the UDP-glucose C6 primary alcohol in two NAD -de- tion and for oxyanion stabilization during formation and break-
pendent steps. The catalytic reaction of UGDH is thought to down of the thiohemiacetal and thioester enzyme intermedi-
involve a Cys nucleophile that promotes formation of a thiohe- ates. Water coordinated to Asp280 deprotonates Cys276 to
miacetal enzyme intermediate in the course of the first oxida- function as an aldehyde trap and also provides oxyanion stabili-
tion step. The thiohemiacetal undergoes further oxidation into zation. Glu161 is the Brønsted base catalytically promoting the
a thioester, and hydrolysis of the thioester completes the cata- thioester hydrolysis.
lytic cycle. Herein we present crystallographic and kinetic evi-
dence for the human form of UGDH that clarifies participation
UDP-glucose 6-dehydrogenase (UGDH; EC 1.1.1.22)2 cata-
lyzes the conversion of UDP-glucose (UDP-Glc) to UDP-glucu-
ronic acid (UDP-GlcUA). The enzyme is responsible for bio-
synthesis of UDP-GlcUA in a multitude of species, including
humans (1). UGDH fuels various cellular metabolic pathways
including the production of extracellular matrix glycosamino-
glycans such as hyaluronan, the synthesis of rare sugars, and
detoxification metabolism (2). Crystal structures of the human
and bacterial forms of UGDH have illustrated the basic anat-
omy of the enzyme (3–7). UGDH folds into oligomers with each
protein subunit composed of two ␣/␤ domains that are con-
nected by a large central ␣-helix. The N-terminal domain is
of covalent catalysis in the enzymatic mechanism. Substitution
of the putative catalytic base for water attack on the thioester
(Glu161) by an incompetent analog (Gln161) gave a UGDH vari-
ant (E161Q) in which the hydrolysis step had become com-
pletely rate-limiting so that a thioester enzyme intermediate
accumulated at steady state. By crystallizing E161Q in the pres-
؉
ence of 5 mM UDP-glucose and 2 mM NAD , we succeeded in
trapping a thiohemiacetal enzyme intermediate and determined
its structure at 2.3 A resolution. Cys276 was covalently modified
˚
in the structure, establishing its role as catalytic nucleophile of
the reaction. The thiohemiacetal reactive C6 was in a position
suitable to become further oxidized by hydride transfer to
ϩ
responsible for binding of NAD , UDP-Glc is bound by inter-
actions from both domains, and the active site is located in the
interdomain cleft. Sequence analysis has shown that elemen-
tary features of UGDH structure and function have been con-
served across the three superkingdoms of life (1). Active-site
residues are essentially invariant among members of the UGDH
enzyme family (1). The homodimeric quarternary structure
adopted by bacterial UGDHs such as that from Streptococcus
pyogenes (SpUGDH) (3) appears to be the basic structural unit
* This work was supported by the Austrian Science Fund (DK Molecular Enzy-
mology W901-B05) and the Structural Genomics Consortium registered
charity (no. 1097737) funded by the Wellcome Trust, GlaxoSmithKline,
Genome Canada, the Canadian Institutes of Health Research, the Ontario
Innovation Trust, the Ontario Research and Development Challenge Fund,
the Canadian Foundation for Innovation, Vinnova, the Swedish Strategic
Research Foundation, the Knut and Alice Wallenberg Foundation, and the
Karolinska Institute. This work was also further supported by the National
Institute for Health Research Oxford Biomedical Research Unit.
Author’s Choice—Final version full access.
□S
This article contains supplemental procedures, Table S1, Figs. S1–S5, and
Scheme S1.
1 To whom correspondence should be addressed: Institute of Biotechnology 2 The abbreviations used are: UGDH, UDP-Glc 6-dehydrogenase; hUGDH,
and Biochemical Engineering, Graz University of Technology, Petersgasse
12/1, A-8010 Graz, Austria. Tel.: 43-316-873-8400; Fax: 43-316-873-8434;
E-mail: bernd.nidetzky@tugraz.at.
UGDH from Homo sapiens; UDP-Glc, UDP-␣-D-glucose; UDP-GlcUA, UDP-␣-
D-glucuronic acid; SpUGDH, UGDH from S. pyogenes; PDB, Protein Data
Bank; GAPN, glyceraldehyde 3-phosphate dehydrogenase.
JANUARY 13, 2012•VOLUME 287•NUMBER 3
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
SCHEME 1. Proposed catalytic mechanism of UGDH, based on the evidence from prior studies of UGDH orthologs from S. pyogenes, bovine liver,
and man.
of UGDH enzyme function. The human UGDH enzyme hUGDH:Cys276) had been replaced by a serine (4, 17). The Cys-
(hUGDH) is arranged into a disc-shaped trimer of such dimers
(4, 5). The more complex oligomeric architecture of hUGDH as
compared with SpUGDH is typical for UGDH from eukaryotic
origin (1, 4, 5) but otherwise does not involve a recognizable
gain of enzymatic function.
to-Ser mutants were almost inactive under standard assay con-
ϩ
ditions. However, their incubation in the presence of NAD
and UDP-Glc led to accumulation of a stable enzyme adduct
that was shown by mass spectrometry (MS) to be derived from
covalent attachment of the oxidized substrate. It was further
demonstrated for SpUGDH (17) that the introduced Ser260 had
become acylated with UDP-GlcUA. Hydrolysis of the unnatural
ester linkage in the C260S mutant was thought to proceed at a
very slow rate, thus allowing accumulation of the intermediate.
Crystal structures of an unreactive ternary complex of wild-
Chemically, the reaction of UGDH is a four-electron oxida-
tion of the C6 alcohol group of UDP-Glc, occurring in two
ϩ
NAD -dependent steps (Equation 1).
UDP-Glc ϩ 2 NADϩ ϩ H2O 3 UDP-GlcUA ϩ 2 NADH ϩ 2 Hϩ
ϩ
(Eq. 1)
type SpUGDH (UDP-xylose/NAD ) and the C260S mutant
(UDP-GlcUA/NADH) showed that Cys260 and Ser260 were
both brought into positions suitable for participation in cova-
lent catalysis (3). In hUGDH ternary complex structures (UDP-
After early elegant studies of the UGDH mechanism per-
formed with bovine liver enzyme (8–16), work of Tanner and
co-workers (3, 17–20) with SpUGDH has led to the current
proposal for the catalytic mechanism (Scheme 1). In this mech-
anism the active-site Cys has the role of a catalytic nucleophile
that intercepts the C6 aldehyde produced in the first step of
substrate oxidation, resulting in formation of a covalent thio-
hemiacetal enzyme intermediate. After replacement of NADH
ϩ
Glc/NADH; UDP-GlcUA/NAD ), Cys276 was likewise placed
appropriately to function as a catalytic nucleophile (4). None-
theless, the proposed covalent intermediates (enzyme bound
thiohemiacetal and thioester species) in the catalytic mecha-
nism of UGDH (Scheme 1) are subject to the caveat of missing
direct affirmation for their formation in the course of the enzy-
matic reaction. Observation of acylenzyme development on
Ser260 in the SpUGDH mutant (17) may not be directly appli-
cable to the situation of the wild-type enzyme where the native
Cys260 offers a substantially different chemical reactivity as
nucleophile or otherwise.
ϩ
by NAD , the reaction proceeds by converting the thiohemi-
acetal into a thioester. Hydrolysis of the thioester enzyme inter-
mediate yields the UDP-GlcUA product.
Recent studies of hUGDH (4, 21, 22) have advanced knowl-
edge of the mechanism (Scheme 1), showing that binding of
ϩ
NAD to an initially formed complex of enzyme and UDP-Glc
A C260A mutant of SpUGDH readily oxidized the chemi-
cally synthesized aldehyde (UDP-gluco-hexodialdose; hydrated
form) to UDP-GlcUA, whereas the same enzyme was inactive
when offered UDP-Glc as substrate. The analogous C276A
mutant of hUGDH was capable of catalyzing the oxidation of
UDP-Glc to UDP-GlcUA, however, at a very low rate (4). Col-
lectively, these findings could be interpreted to mean that the
second oxidation step of the UGDH reaction might occur even
in the absence of a catalytic nucleophile. In contrast, Ridley et
al. (11) showed that a cyanide-derivatized form of bovine liver
UGDH was even more active than the unmodified enzyme in
reduction of UDP-gluco-hexodialdose, whereas only the
is accompanied by a large rigid-body movement of the N-ter-
minal domain, resulting in seclusion of substrate and coenzyme
in their reactive positions through interdomain closure. Coen-
zyme exchange at the stage of thiohemiacetal enzyme interme-
diate, therefore, necessitates a full cycle of domain opening
ϩ
(release of NADH) and closing (binding of NAD ), and this
overall step was shown to be rate-determining for the human
enzyme reaction in the presence of physiological concentra-
ϩ
tions of NAD .
Useful evidence in support of covalent catalysis by UGDH
was obtained from the characterization of site-directed
mutants in which the candidate nucleophile (SpUGDH:Cys260
2120 JOURNAL OF BIOLOGICAL CHEMISTRY
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
TABLE 1
unmodified enzyme was reactive in the oxidation of the same
Crystallographic data collection and refinement statistics
E161Q mutant (thiohemiacetal intermediate)
Data collection
substrate. It becomes apparent, therefore, that covalent cataly-
sis by cysteine is not fundamentally well understood in its
molecular basis, and despite the significant research efforts in
different groups, the catalytic mechanism of UGDH remains
elusive.
PDB accession code
Synchrotron beamline
Wavelength (Å)
3khu
Diamond I03
0.9763
Space group
P21212
We wish to communicate herein a study of hUGDH that
provides clarification. Using site-directed replacement of the
presumed catalytic base for hydrolysis of thioester (Glu161) by
an incompetent analog (Gln), we found that the rate of the
hydrolysis step had become selectively reduced in the E161Q
mutant, thus allowing buildup of a thioester enzyme interme-
diate at steady state. By crystallizing the E161Q mutant under
Unit cell dimensions
a ϭ 203.5, b ϭ 207.3,
c ϭ 93.4 Å
␣ ϭ ␤ ϭ ␥ ϭ 90.0°
48.74–2.30 (2.42–2.30)
Resolution rangea (Å)
No. unique reflectionsa 175,128 (25,412)
Completenessa (%)
99.8 (100.0)
9.4 (2.0)
a
͗I/␴(I)͘
a
Rmerge
0.106 (0.743)
4.3 (4.3)
Redundancya
ϩ
Refinement
conditions of a limiting concentration of NAD while UDP-
No. Atoms P/L/Ob
21,866/432/1397
0.195/0.225
glucose was present in saturating amounts, we succeeded in
intercepting a thiohemiacetal enzyme intermediate in the crys-
tal and determined its structure at 2.3 Å resolution. With this
structure the mechanism of covalent catalysis by Cys276 can be
settled. However, the structure of the E161Q intermediate has
two additional aspects of relevance. First, it shows that the pro-
tein subunit adopts an apoenzyme-like “open domain” confor-
R
work/Rfree
r.m.s.d. bond lengthc (Å) 0.016
r.m.s.d. bond anglec (°) 1.438
B
mean P/L/Ob (Å2)
23.9/42.9/27.5
a Values in parentheses show the statistics for the highest resolution shells.
b P/L/O represents protein/ligand/other (water, ion, and solvent).
c r.m.s.d. indicates root mean square deviation.
mation and does not contain a NAD(H) ligand at the coenzyme N-terminal extension that harbors a solubility enhancement
binding site, indicating that the mutant has actually undergone tag, a streptavidin tag, and a tobacco etch virus protease cleav-
intermittent domain opening to allow for the release of NADH age site. Enzymes were purified using a previously described
from the thiohemiacetal enzyme intermediate, as proposed three-step procedure consisting of affinity chromatography, gel
recently (4). Second, there is appreciable interest in the devel- filtration (Superdex 200 16/60 HiLoad, GE Healthcare), and
opment of antagonists of hUGDH (23, 24). The relevant form of anion exchange chromatography (HiTrap-Q HP, GE Health-
the enzyme to be targeted with reversible inhibitors is the thio- care) (4). The N-terminal extension was removed before gel
hemiacetal intermediate, which accumulates at the steady state filtration using tobacco etch virus protease. Details of the pro-
(4). By studying mutants of two other prominent residues in the cedures are given elsewhere (4). All mutants were shown by gel
active site of hUGDH (Lys220 and Asp280; Refs. 1, 21, and 22), filtration analysis to form hexamers in solution, as does native
we have also delineated the steps in catalysis that lead up to the hUGDH.
thiohemiacetal intermediate. Finally, comparison of wild-type
Crystallization of E161Q, Data Collection, and Refinement—
ϩ
enzyme and E161Q in pH studies gave clear evidence in support The E161Q protein solution containing 2 mM NAD and 5 mM
of a general base catalytic function of Glu161 in the hydrolysis UDP-Glc was concentrated to 15 mg/ml (ϳ0.3 mM). Crystals
step of the reaction. For the first time, therefore, a comprehen- were grown at 4 °C in 150-nl sitting drops, equilibrated against
sive catalytic mechanism for hUGDH is proposed.
mother liquor containing 18% PEG “smears” (a mixture of ten
PEG polymers with molecular masses ranging from 400 to
10000 Da), 5% ethylene glycol, and 0.1 M HEPES (pH 7.5). Dif-
EXPERIMENTAL PROCEDURES
Materials—Unless stated otherwise, all materials were of the fraction data were collected at 100 K at Diamond beamline I03.
ϩ
highest purity available from Sigma. NAD was obtained from Data were processed with MOSFLM (25, 26) and subsequently
Roth (Karlsruhe, Germany) in a purity of Ͼ98%.
scaled using the program SCALA (25). The structure of E161Q
Site-directed Mutagenesis and Preparation of Enzymes—The was solved by molecular replacement using the Phaser (27) pro-
enzyme referred to as native or wild-type hUGDH is a trun- gram and the structure of hUGDH (PDB code 2q3e) as the
cated version of the natural enzyme that lacks 27 amino acids search model. The structure was manually rebuilt in COOT
from the C terminus. It was previously shown that the trunca- (28), and restrained refinement with appropriate TLS groups
tion neither affects the enzyme activity nor the homohexameric was performed using REFMAC5 (29). Data collection and
oligomerization of hUGDH in solution (4). Variants of hUGDH refinement statistics are summarized in Table 1.
likewise contain the C-terminal truncation. Mutagenesis lead-
Enzyme Kinetics—Reactions were performed at 25 (Ϯ 1) °C in
ing to site-directed substitution of Glu161 by Gln (E161Q), potassium phosphate buffer (50 mM; pH 7.5). Initial rates were
Lys220 by Ala (K220A), Asp280 by Ala (D280A), Thr131 by Ser obtained by measuring the absorbance of NADH produced at
(T131S), and Asn224 by Ala (N224A) was performed employing 340 nm (⑀NADH ϭ 6220 MϪ1 cmϪ1). The duration of the reac-
the Stratagene QuikChange site-directed mutagenesis kit tion was between 10 and 350 min, depending on the activity of
according to standard protocols and using the wild-type gene as the enzyme used. Unless mentioned otherwise, initial rates
template. The oligonucleotide primers used are listed in the were obtained under conditions in which the applied enzyme
supplemental procedures. The mutants were recombinantly had undergone at least three turnovers. Measurements were
produced in Escherichia coli BL21(DE3)-R3 using a pBEN-de- performed in triplicate, and the data were averaged. Kinetic
rived plasmid vector, encoding the target protein fused to an parameters (Vmax, Km) were calculated from initial-rate data
JANUARY 13, 2012•VOLUME 287•NUMBER 3
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
ϩ
recorded at varied concentrations of UDP-Glc or NAD using
TABLE 2
Kinetic parameters for the E161Q mutant, determined at 25 °C and pH
7.5, in comparison to wild-type hUGDH
a constant saturating concentration of the respective other sub-
ϩ
strate (15 mM NAD , 4 mM UDP-Glc). Turnover numbers (kcat
)
Wild typea
E161Q
1
were calculated by dividing ⁄
2V
max (2 NADH produced/1 UDP-
Ϫ1
k
cat (s
)
0.85 Ϯ 0.1
0.7 Ϯ 0.1
35 Ϯ 5
1.2
0.0014 Ϯ 0.0001
0.10 Ϯ 0.01
55 Ϯ 10
Glc converted) with the molar concentration of enzyme active
sites (E). The protein concentration was determined from the
absorbance at 280 nm using a molar extinction coefficient of
K
mC (mM)
K
mS (␮M)
Ϫ1
k
k
k
cat /KmC (sϪ1mM
)
0.014
cat/KmS (sϪ1mM
)
24
0.025
Ϫ1
Ϫ1 b
48360 MϪ1cmϪ1 (30). KmS is the Km for UDP-Glc substrate, and
obs (s
)
ϳ10
0.33 Ϯ 0.01
0.16 Ϯ 0.02
1.0 Ϯ 0.1
2.1
K
dC (mM)c
0.4
ϩ
K
mC is the Km for NAD coenzyme.
b
⌸/[E] ͓Ϫ͔
0.3
Ϫ1
k
obs/KdC (sϪ1mM
)
25
The E161Q mutant was characterized in stopped-flow
a Data are from Egger et al. (4).
kinetic experiments performed according to procedures
described elsewhere (4). Reaction time courses were recorded
in triplicate and averaged. The limiting concentration of
enzyme active sites was in the range 10–15 ␮M. A solution of
E161Q saturated with UDP-Glc (1 mM) was mixed with a solu-
b UDP-Glc (1 mM) and NAD (15 mM) were present at saturating concentrations.
ϩ
c Value was obtained from non-linear fits of kobs vs. ͓NAD ͔ data with a single
ϩ
rectangular hyperbola function.
obst
[NADH]t ϭ ⌸͑1 Ϫ eϪk ͒ ϩ Vsst ϩ Y0
(Eq. 2)
ϩ
tion containing UDP-Glc (1 mM) and NAD (0.2–20 mM). Suit-
log Y ϭ log [C/͑1 ϩ [Hϩ]/K͒]
(Eq. 3)
(Eq. 4)
able controls were obtained by mixing reaction solutions lack-
ing the enzyme. Apparent first-order rate constants (kobs) for
the kinetic transient were obtained from an appropriate fit of
the stopped-flow progress curves, as described under “Data
Analysis” below.
log Y ϭ log [(CH ϩ CL͑[Hϩ]/K͒/͑1 ϩ ͓Hϩ]/K͒͒]
Mass Spectrometry Analysis—The electrospray ionization-
TOF-MS method applied to identify covalent adduct formation
in E161Q was described in a previous publication (4).
Functional complementation of K220A by externally added
primary amines was examined by comparing initial rates mea-
sured in the absence and presence of the amine whose concen-
tration was varied in the range 10–500 mM. Experiments were
performed at pH values from 6.5 to 9.0. A similar analysis was
carried out with D280A studying the effect of external anions
on the activity of the mutant (pH 7.5). Chloride, bromide, azide,
formate, and acetate were tested as their sodium salts in a con-
centration range between 10 and 300 mM.
RESULTS AND DISCUSSION
Kinetic Characterization of E161Q Mutant—Crystal struc-
tures of hUGDH in the apo form (PDB code 3itk) and in a
ternary complex with UDP-Glc and NADH (PDB code 2q3e)
ϩ
have shown that to bring the nicotinamide ring of NAD into
hydride transfer position Glu161 must move away from its orig-
inal place in the active site of the apoenzyme (4). In the reactive
conformation of the enzyme, which we believe is represented
by the structure of the hUGDHꢀUDP-Glc/NADH complex,
Glu161 is drawn away from the nicotinamide by contact with
Lys129. A third structure of hUGH in complex with the UDP-
pH Studies—The pH dependences of steady-state kinetic
parameters kcat and kcat/KmC were determined in the pH range
5.0–10.5 for wild-type hUGDH. A three-component buffer,
composed of MES, Tris, and glycine, was used that displayed a
pH-independent ionic strength of 0.1 M. The pH dependence of
the apparent kcat for E161Q was determined in the pH range
6.0–9.0. The apparent kcat was obtained from an initial-rate
measurement at saturating concentrations of UDP-Glc (5 mM)
ϩ
GlcUA product and NAD (PDB code 2qg4) revealed that
Glu161 was conformationally flexible. In half of the enzyme sub-
units the side chain of Glu161 adopted the position inside the
active site, resulting in a displacement of the nicotinamide por-
tion of the coenzyme. In the remainder of the subunits, Glu161
was in the alternative “out” conformation, and an ordered nic-
otinamide moiety was observed. This conformational flexibility
of Glu161 distinguished the product structure from the struc-
ture of the substrate complex, where Glu161 was found exclu-
sively in the out conformation. Based on this structural evi-
dence, we speculated that Glu161 could serve the role of a
general base in the catalytic mechanism, assisting the attack of
water on the putative thioester intermediate. Therefore, Glu161
was a very interesting target for site-directed mutagenesis, and
it was replaced by Gln with the aim of disrupting the proposed
hydrolysis step.
ϩ
and NAD (15 mM). The pH dependence of kobs for the wild-
type enzyme was obtained in the pH range 6.0–8.5. The con-
centration of UDP-Glc was 1.0 mM and at each pH kobs was
ϩ
determined at two concentrations of NAD that were either
limiting (0.1 mM; ϽKmC) or saturating at the steady state (10
mM).
Data Analysis—Sigma Plot 2004 software (Version 9.01) was
used for nonlinear least squares regression analysis. Equation 2
describes NADH formation in multiple turnover stopped flow
experiments. [NADH]t is the concentration of NADH at time t,
⌸ is the [NADH] amplitude of the kinetic transient, Vss is the
steady-state rate, and Y0 stands for the (absorbance) back-
ground of the control. Reactions start at Y0. Equation 3
The purified E161Q mutant displayed a 610-fold decrease in
k
cat as compared with wild-type hUGDH (Table 2 summarizes
describes a pH dependence where activity (Y ϭ kcat, kcat/KmC
,
results from steady-state and presteady-state kinetics). The cat-
ϩ
k
obs) is constant at high pH and decreases below apparent pK. C
alytic efficiencies for UDP-Glc (kcat/KmS) and NAD (kcat
/
is the pH-independent value of Y at the optimum state of pro-
tonation. Equation 4 describes a sigmoidal pH dependence with
constant values of Y at high (CH) and low pH (CL), and an
inflection point at pK.
K
mC) were also strongly affected by the mutation. These results
indicate that Glu161 has an important role for the enzymatic
function of hUGDH, and its substitution by Gln affects the cat-
alytic reaction at different steps. The proposed kinetic mecha-
2122 JOURNAL OF BIOLOGICAL CHEMISTRY
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
FIGURE 1. Kinetic and mass spectrometric evidence for stable intermediate accumulation in E161Q. A, shown are typical stopped-flow progress curves of
the enzymatic reaction obtained at 10 mM (I) or (125 ␮M (II) NADϩ and 1 mM UDP-Glc using 15 ␮M enzyme subunits. The gray lines are the fit of Equation 1 to the
data. A respective reference measurement lacking the enzyme is also shown. B, shown is dependence of burst magnitude on the concentration of NADϩ used
in the reaction for E161Q (filled circles) and wild-type enzyme (open circles; data are from Ref. 4). C, shown are deconvoluted data from ESI-MS characterization
of a protein sample obtained after a 1-min reaction time from a conversion experiment, performed using 20 ␮M E161Q, 15 mM NADϩ, and 1 mM UDP-Glc. The
left peak corresponds to the underivatized mutant protein. The right peak shows a mass increase of 562 g/mol, consistent with formation of a thioester enzyme
intermediate.
ϩ
reasonable to assume that the NAD -dependent oxidation
nism of hUGDH (supplemental Scheme S1) implies that kcat
/
K
mS is a direct measure of UDP-Glc binding to the free enzyme steps of the reaction, not the hydrolysis step, become acceler-
ϩ
(k1), whereas kcat/KmC is a complex parameter that includes all ated in response to increasing levels of NAD . Consequently,
ϩ
steps from binding of NAD to enzymeꢀUDP-Glc (k3) up to the breakdown of the thioester intermediate is strongly sug-
dissociation of NADH from the thioester intermediate (k9). The gested to be rate-determining for the E161Q reaction at steady
kcat involves all unimolecular steps of the mechanism (k5, k9, state. Of note, the wild-type enzyme shows a dependence of
ϩ
k11, k13). Stopped-flow experiments were, therefore, performed ⌸/[E] on NAD concentration that is completely opposite that
with E161Q to resolve kinetic complexity of the steady-state of the E161Q mutant (Fig. 1B), with a marked decline of ⌸/[E]
ϩ
parameters and to analyze the different parts of the enzymatic at high NAD , suggesting overall rate limitation at the step of
reaction (Scheme 1) as kinetically separate steps. The time aldehyde oxidation.
course of UDP-Glc conversion by E161Q showed a large tran-
The value of kobs associated with the kinetic transient for the
ϩ
sient “burst” of NADH formation that was followed by the reaction of E161Q at saturating concentration of NAD was
much slower steady-state phase (Fig. 1A). A fit of the data with 0.33 sϪ1, which is just 30-fold decreased as compared with the
Ϫ1
Equation 2 provided estimates for kobs and the burst amplitude, corresponding value of kobs for the wild-type enzyme (10 s
;
⌸. Reactions were performed at different concentrations of Table 2). Therefore, this shows that the disruptive effect of the
NAD , and we show in Fig. 1B that the molar ratio of ⌸ and [E] Glu161 3 Gln mutation was ϳ20 times stronger on the steady-
ϩ
(concentration of enzyme subunits used in the reaction) state constant (kcat) than it was on the transient rate constant
ϩ
ϩ
increased with increased NAD concentration and that it (kobs). From the hyperbolic dependence of kobs on NAD , we
ϩ
approached a value of 1 at high coenzyme levels. This result determined an apparent dissociation constant of NAD from
implies that there is a slow step in the enzymatic reaction of the enzymeꢀUDP-Glc (KdC) and show in Table 2 that the ratio kobs
/
E161Q mutant that occurs after the formation of NADH,
KdC was just 12-fold decreased in E161Q as compared with the
whether the NADH was derived from both oxidation steps in wild-type enzyme. In wild-type hUGDH, kobs/KdC stands for
the case that hydrolysis was rate-limiting or only from the first the first oxidation step of the reaction (supplemental Scheme
step of alcohol oxidation in the case that aldehyde oxidation S1). The implication of these findings that substitution of
was rate-limiting. The contribution of this slow step to overall Glu161 by Gln appears to have selectively decelerated one or
ϩ
rate limitation strongly increases when the NAD concentra- more of the reaction steps coming after alcohol oxidation
tion is raised, as evident from Fig. 1B. The catalytic step of immediately led to the possibility that a relatively stable inter-
ϩ
thioester hydrolysis lacks NAD dependence, so it is therefore mediate could be formed in E161Q under conditions when
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
FIGURE 2. Structural characterization of the E161Q mutant. A, shown is structural superimposition of the homohexamers of E161Q and wild-type hUGDH
bound to UDP-Glc and NADH. B, shown is superimposition of the protein subunits of E161Q (gray schematic, UDP-Glc molecules are shown) and wild-type
hUGDH (ternary complex with UDP-Glc and NADH (red schematic, closed conformation) and apo structure (green schematic, open conformation)). Note that
E161Qadoptsaquasi-openconformation. C, shownisaclose-upstereoviewoftheactivesiteofthethiohemiacetalenzymeintermediateintheE161Qmutant.
The electron density map (2Fo Ϫ Fc, contoured at 1.0 ␴) illustrates the presence of the covalent intermediate. Conformation states of Gln161 in the in and out
positions are shown. Water molecules are shown as red spheres, and hydrogen bonding interactions (Յ3.1 Å) are depicted by dashed lines. D, shown is
superimpositionofthecovalentlylinkedsubstrateinE161Q(white, C-atoms)withUDP-Glc(green, C-atoms)andUDP-GlcUA(blue, C-atoms)boundbywild-type
enzyme. The NAD(H) present in the complexes of wild-type enzyme is also shown. The distances between the reactive carbons of substrate (glucosyl C6) and
the nicotinamide ring of coenzyme (C4) are shown. The distance is favorable for hydride transfer, suggesting that the thiohemiacetal is bound in a position
suitable to become further oxidized by NADϩ.
UDP-Glc is saturating. This was examined by a time-resolved of instability of the covalent intermediate under the condi-
MS experiment.
tions of MS analysis.
Upon incubation of E161Q (20 ␮M) in the presence of satu-
Crystal Structure of E161Q Mutant Shows Trapped Thiohe-
ϩ
rating concentrations of UDP-Glc (1 mM) and NAD (15 mM), miacetal Enzyme Intermediate—E161Q was crystallized in the
we identified a macromolecular species which accumulated presence of UDP-Glc and NAD , and the structure was deter-
ϩ
that displayed a ϩ562 Ϯ 0.1 g/mol mass increase relative to mined at 2.3 Å resolution (Table 1; PDB code 3khu). The overall
apoenzyme. The expected mass increase would be ϩ562 g/mol homohexamer structure of the mutant is highly similar to the
if a catalytically competent (thio)ester intermediate of E161Q previously reported structures of hUGDH (Fig. 2A). In each
was accumulated in the steady-state phase of substrate conver- subunit of E161Q (Fig. 2B), the substrate binding site was occu-
sion (⌬mass: ϩ564 g/mol for hemiacetal). Deconvolution of pied by UDP-Glc. Interestingly, the coenzyme binding site in
ϩ
the MS data showed two peaks of similar size, hence presum- the N-terminal domain did not contain NAD , but it had UDP-
ϩ
able abundance: one corresponding to native E161Q and Glc bound instead. Anchoring of UDP-Glc as the NAD surro-
another corresponding to the enzyme intermediate (Fig. 1C). gate occurred mainly through its UDP moiety, mimicking the
ϩ
The observed ⌸/[E] ratio of 1 for the mutant (Fig. 1B) is thus adenine portion of the NAD . The corresponding glucosyl
best consistent with an acylenzyme intermediate of E161Q moiety appears to have been bound comparatively weakly,
produced in two successive oxidation steps of UDP-Glc. The exploiting interactions completely different from the ones used
ϩ
relative abundance of the covalent intermediate of E161Q for productive binding of the nicotinamide ribose of NAD (see
was a maximum at steady state (ϳ1 min) and decreased upon later in text).
prolonged incubation, reflecting the progressing depletion
Assessment of the electron density in the active sites of all six
of the limiting UDP-Glc substrate (supplemental Fig. S1). molecules in the asymmetric unit of the E161Q structure
Therefore, this indicates that the presence of unmodified revealed a clear additional, incessant density bridging the UDP-
enzyme in the analyzed samples was a true characteristic of Glc C6 and Cys276 S␥ atoms, as shown in Fig. 2C. The distance
the catalytic reaction in solution and did not occur as a result between the carbon and sulfur atoms was Ͻ2 Å, where for a
2124 JOURNAL OF BIOLOGICAL CHEMISTRY
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
non-bonded interaction the atoms would be expected to have a
distance of Ͼ3.5 Å (31) (the sum of the van der Waals radii of
a carbon and sulfur atom). Therefore, this confirmed the pres-
ence of a covalent intermediate on Cys276. Supplemental Fig. S2
provides a stereo depiction of the covalent intermediate.
Because the O6 atom of the sugar ring is not coplanar with the
C6-S␥ bond, the electron density (Fig. 2C) clearly indicates an
sp3-hybridized carbon at the reactive center of the substrate,
implying that a tetrahedral thiohemiacetal (not a thioester)
intermediate was trapped in the E161Q structure.3 Limited
ϩ
NAD (2 mM) relative to UDP-Glc (5 mM) plus competition
by UDP-Glc for binding to the coenzyme binding site could
explain why the E161Q reaction stopped after one round of
oxidation in the crystal. Moreover, the low temperature used
and high solution viscosity could be additional factors.
Structural comparison of the E161Q intermediate to the
wild-type hUGDH bound with UDP-Glc/NADH and UDP-
ϩ
GlcUA/NAD shows that the spatial arrangement of residues
in the immediate vicinity of the substrate reactive C6 group was
hardly affected by formation of the intermediate (Fig. 2D; sup-
plemental Fig. S3). The notable exception is the side chain of
Cys276, which has undergone an ϳ35° rotation in the interme-
diate structure as compared with the ternary complex struc-
tures, displacing the S␥ atom by 2 Å. The position of the gluco-
pyranosyl ring in the substrate binding pocket was unchanged
FIGURE 3. Time course analysis for reactions of D280A (I) and K220A (II)
mutantsofhUGDH. Inset, FunctionalcomplementationofK220Awithmeth-
ylamine (100 mM) stimulates the reaction rate. The concentrations of UDP-Glc
and NADϩ were 1 and 15 mM, respectively. The molar enzyme concentration
was 100 ␮M. Fits of the appropriate equation to experimental data are shown
as gray lines.
in the intermediate structure as compared with the ternary the “fully open” conformation seen in the apoenzyme (Fig. 2B).
complex structures. Gln161 is seen to adopt two different con- We interpret this structural feature of E161Q to imply that the
formations in and out of the active site, as shown in Fig. 2C. A mutant has undergone intermittent domain opening to allow
structural overlay of the ternary complexes and the thiohemi- for the release of the NADH produced during formation of the
acetal intermediate (having Gln161 in the out conformation) thiohemiacetal enzyme intermediate. Attachment of UDP-Glc
ϩ
indicates that the C6 of covalently bound UDP-gluco-hexodial- to the site where NAD normally binds eventually traps the
ϩ
dose would be within suitable hydride transfer distance (2.5– intermediate in the crystal, and unlike binding of NAD , UDP-
2.8 Å) to the nicotinamide C4 (Fig. 2D), suggesting that a cata- Glc appears to be not sufficient to promote the complete “clos-
lytically competent intermediate has been trapped in the ing” motion of the N-terminal domain.
crystal.
Roles of Lys220 and Asp280 in Covalent Catalysis by hUGDH—
Fig. 2B displays a structural superimposition of the protein The function of Cys276 as catalytic nucleophile of the reaction
subunits of the E161Q intermediate, the native hUGDH in of hUGDH is validated by crystallographic and kinetic evidence
complex with UDP-Glc/NADH and the apoT131A structure. It on the E161Q mutant (Figs. 1 and 2). However, there is the
was previously shown that upon anchoring of UDP-Glc and mechanistically relevant question of participation of the other
NADH to the apoenzyme, the hUGDH subunit undergoes a active-site residues in providing catalytic assistance for cova-
large “domain closure” conformational change that can be best lent intermediate formation. The structure of hUGDH was pre-
envisioned as an ϳ13° rotation of the N-terminal domain viously interpreted to support a role of Lys220 as the catalytic
toward the assumed immobile C-terminal domain (4). The ␣10 base for abstraction of alcohol proton in the first oxidation step.
helix was identified as the hinge region for this rotation. It is An active-site water bonded to Asp280 was suggested to depro-
very interesting that the E161Q intermediate now adopts a tonate the thiol side chain of Cys276 for nucleophilic attack.
“quasi open” domain orientation that is only 3° different from However, one caveat of the mechanistic proposal is that sup-
porting biochemical evidence was previously lacking. We,
therefore, substituted Lys220 and Asp280 individually by Ala and
3 It is interesting to compare the structure of the thiohemiacetal intermediate
performed a kinetic characterization of purified K220A and
of E161Q to the structure of a thioacylenzyme intermediate of a E268A
D280A mutants. Both mutants showed extremely low levels of
mutant of glyceraldehyde 3-phosphate dehydrogenase (GAPN) from
Streptococcusmutans(32). JustlikeGlu161 inhUGDH, thesubstitutedGlu268
of GAPN was the assumed catalytic base for thioester hydrolysis. The crys-
UGDH activity, consistent with previous findings of Simpson
and co-workers (21, 22). Just 75% of a full turnover was
talstructureofthetrappedintermediateoftheGAPNmutantwassolvedat
observed in ϳ5 h (Fig. 3), assuming that all active sites pres-
a resolution (2.55 Å) similar to that obtained here for the E161Q mutant
ent in the assay undergo turnover at the same time and form
structure. The flat density shape at C1 of the covalent adduct of GAPN was
consistent with the sp2 hybridization expected for a thioacylenzyme inter-
two NADHs during the reaction. Despite these limitations in
mediate. WerefertothestudyofD’Ambrosioetal. onGAPN(32)toempha-
size that the crystallographic data on the covalent intermediate of E161Q
do allow unambiguous distinction between an enzyme-bound hemiacetal
and ester.
terms of steady-state kinetic analysis, the data could be used
to derive an estimate for kobs, calculated from observed
NADH rate (D280A) or by fitting data with Equation 2
JANUARY 13, 2012•VOLUME 287•NUMBER 3
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
FIGURE 4. pH profile analysis for wild-type hUGDH and E161Q mutant. A, steady-state kinetic data show kcat for wild-type enzyme (filled circles) and E161Q
(down triangles) and kcat/ KmC (open circles) for wild-type enzyme. B, transient kinetic data were obtained for wild-type hUGDH showing kobs (open symbols) and
⌸/[E] (closed symbols) recorded at high (10 mM; circles) and low (0.1 mM; triangles) concentration of NADϩ. UDP-Glc was used in a concentration of 1 mM. The
symbols show the experimental data, and solid lines are fits with the suitable equation.
(K220A). The kobs values thus obtained were decreased by removal by the water molecule coordinated to Asp280. The
about 5 orders of magnitude in comparison to kobs of the observed impairment of the D280A mutant can thus proba-
wild-type enzyme (supplemental Table S1). The kobs for bly be explained by loss of participation from covalent catal-
ϩ
D280A displayed a linear dependence on the NAD concentra- ysis in the alcohol dehydrogenase step of the reaction. In
tion up to 10 mM. The lowered coenzyme binding affinity of accordance with this notion, both character and magnitude
D280A compared with wild-type enzyme can probably be of the disruptive effect on the enzymatic reaction that
explained by disruption of a water-mediated interaction resulted from complete deletion of the catalytic nucleophile
ϩ
between the side chain of Asp280 and the NAD ribose 2-OH, in a C276A mutant of hUGDH (4) were highly similar to the
observed in the structure of the enzyme complex with UDP-Glc just-described kinetic consequences of the Asp280 3 Ala
and NADH (4).
replacement. Note: the activity of D280A was not rescued by
In the Fig. 3 inset we show that external methylamine elicited the addition of various anions (chloride, bromide, azide, for-
functional complementation of K220A, giving a substantial mate, or acetate).
(kobs, ϳ20-fold; apparent kcat, ϳ120-fold) rate enhancement in
Besides Lys220, Cys276, and Asp280, there are two other con-
the conversion of UDP-Glc by the mutant (supplemental Table served residues in the active site of hUGDH: Thr131 and Asn224
S1). The “chemical rescue” of K220A displayed a saturable (Fig. 2C). Their importance for UGDH activity was examined in
dependence on the methylamine concentration (supplemental T131S and N224A mutants. Steady-state kinetic parameters of
Table S1), suggesting that the mutant enzyme is capable of the two mutants were within an order of magnitude of the
binding the amine, presumably at the site vacated by substitu- native enzyme (supplemental Table S1), suggesting that neither
tion of Lys220 with Ala, and that the enzyme-amine complex is residue is essential for catalysis by hUGDH.
more active than the free K220A enzyme. We also showed that
Role of Glu161 as Catalytic Base for Thioester Hydrolysis,
complementation of K220A by methylamine worked best at Revealed in pH Studies—So far we have presented evidence that
high pH (supplemental Fig. S4), as expected if the unproto- emphasized the importance of Glu161 in the catalytic step of
nated form of the amine was required for activity enhance- thioester hydrolysis. To show the exact role of the Glu in this
ment, which is consistent with participation of the unproto- step, we analyzed the pH dependences of the reaction rate con-
nated ⑀-NH2 group of Lys220 in catalysis. In summary, these stants for wild-type enzyme and E161Q. Experimental data and
results are consistent with the proposed base catalytic func- their fits with a suitable mathematical model are shown in Fig.
tion of Lys220 during the alcohol oxidation step of the reac- 4. Table 3 summarizes the apparent ionization constants deter-
tion. They also suggest an essential role of the lysine, likely as mined from the pH profiles. The kcat for wild-type hUGDH was
oxyanion stabilizer, during the second oxidation and thioes- constant at high pH and decreased below a pK of 7.0 (Ϯ0.1).
ϩ
ter hydrolysis.
The kobs determined at high concentration of NAD displayed
The kinetic behavior of the D280A mutant (Fig. 3) is inter- essentially the same pH dependence as kcat (pK ϭ 7.1 Ϯ 0.1).
ϩ
preted to support that catalysis in the first oxidation step of The pH dependence of kobs determined at low NAD revealed
hUGDH has become massively disrupted as a result of the a substantially lower pK of 6.6 (Ϯ 0.3). The observation that kobs
mutation. This finding fits very well with the idea described of the wild-type enzyme is governed by a different apparent
ϩ
in our previous paper on hUGDH that oxidation of UDP-Glc ionization depending on the level of NAD used in the reaction
ϩ
by hydride transfer to NAD is strongly coupled to forma- suggests that the rate-limiting step for kobs changes in response
ϩ
tion of the thiohemiacetal enzyme intermediate (4). To per- to variation of the NAD concentration. This result is consis-
form nucleophilic attack on the incipient aldehyde, the side tent with findings from our previous paper on hUGDH, that
chain of Cys276 needs to be activated by deprotonation, and showed that decreasing the NAD concentration results in a
ϩ
this was thought to occur through just-in-time proton gradual increase in the contribution of the second oxidation
2126 JOURNAL OF BIOLOGICAL CHEMISTRY
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
step to the overall rate limitation, which is in kcat. It was specif- base catalysis by water/hydroxide ion in the mutant. The pro-
ϩ
posed function of Glu161 implies that protonation of its side
chain below its apparent pK should make the catalytic step of
thioester hydrolysis progressively more rate-limiting for the
kcat of the wild-type enzyme, observable as an increase in the
burst magnitude ⌸/[E] upon lowering the pH. One can see in
Fig. 4B that ⌸/[E] indeed has the predicted pH dependence,
increasing from a low constant value at high pH to a higher,
also constant value at low pH. The maximum value of ⌸/[E]
ically proposed that exchange of NADH by NAD progres-
sively becomes the slowest step of the reaction under these
ϩ
conditions, implying that the kobs determined at low NAD
includes only steps of the reaction up to the thiohemiacetal
ϩ
intermediate, ready to bind new NAD . It was revealing to
observe, therefore, that the steady-state rate constant (Vss/E)
obtained from stopped-flow progress curves measured at sub-
ϩ
K
mC levels of NAD displayed a pH dependence similar to the
ϩ
at high NAD at pH 6.0, where Glu161 should be largely
pH dependence of kcat (pK ϭ 7.6 Ϯ 0.2). The pH-dependent
step(s) that controls the pH profile of kcat for the wild-type
enzyme must, therefore, be located in the steps used for the
second oxidation and hydrolysis.
protonated, corresponds to about half the molar equivalent
of enzyme active sites present in the reaction. Of note, the
pH dependence of ⌸/[E] was only seen under conditions of a
ϩ
The kcat for E161Q increased linearly (slope: 0.41 Ϯ 0.03) as
the pH was raised from 6.0 to 9.0 (Fig. 4A). The apparent ioni-
zation of pK ϭ 7.0 present in the pH-log kcat profile of native
hUGDH was clearly eliminated from the pH dependence of the
mutant. The pH dependence of E161Q conforms with expecta-
tion for the consequence of removal of a general base catalytic
group from the enzyme and is consistent with a role for specific
high concentration of NAD . Using a low concentration of
ϩ
NAD to make the second oxidation rate-limiting, ⌸/[E]
was independent of pH, as expected because Glu161 is not a
candidate catalytic residue in this step. In summary, there-
fore, the evidence from the pH studies strongly supports a
role of Glu161 as Brønsted catalytic base for hydrolysis of the
thioester enzyme intermediate.
The structure of the E161Q thiohemiacetal intermediate
provides a very interesting detail about the possible mechanism
of thioester hydrolysis, showing that a water molecule bonded
to Gln161 (3.6 Å) would be in a suitable position to potentially
serve as incoming nucleophile for the reaction (Fig. 2C).
Assuming that a similar arrangement of groups occurs at the
level of the thioester intermediate, there is the highly attrac-
tive feature that hydrolysis could be achieved through a tet-
rahedral transition state geometrically analogous to the ani-
onic thiohemiacetal, thus allowing full utilization of the
oxyanion-stabilizing capabilities of the hUGDH catalytic
center in all three steps of a parsimonious reaction coordi-
nate, summarized in Scheme 2. Although the water coordi-
nated to Asp280 has been proposed to be involved in hydro-
lysis (33), our data indicate that this water is unlikely to
participate as reactant in the hydrolysis step. The proposed
role of the mobile Glu161 in this mechanism involves dis-
TABLE 3
Summary of results of pH studies
wild-type hUGDH
logY
pK
C
r2 Equation
Steady state
Ϫ1
kcat
7.0 Ϯ 0.1
1.1 Ϯ 0.1 s
0.99
3
3
Ϫ1
kcat/KmC
7.7 Ϯ 0.2
2715 Ϯ 461 MϪ1s
0.98
Presteady state
ϩ
10 mM NAD
kobs
Ϫ1
Ϫ1
7.1 Ϯ 0.1
7.3 Ϯ 0.1
6.5 Ϯ 0.3
15.6 Ϯ 1.5 s
0.99
0.99
0.99
3
3
4
Vss
1.5 Ϯ 0.1 s
⌸/͓E͔
CH ϭ 0.12 Ϯ 0.01
CL ϭ 0.5 Ϯ 0.1
ϩ
100 ␮M NAD
kobs
Ϫ1
6.6 Ϯ 0.3
7.6 Ϯ 0.2
No
6 Ϯ 1 s
0.88
0.99
3
3
Ϫ1
Vss
0.4 Ϯ 0.1 s
⌸/͓E͔
0.21 Ϯ 0.05a
E161Q
kcat
slope: 0.41 Ϯ 0.03
0.98 LRb
a Average and S.D. of ⌸/͓E͔ obtained in the pH range 6.0–8.5 (see Fig. 4B).
b LR, linear regression.
SCHEME 2. Panel A, the proposed mechanism of hydrolysis of the thioacylenzyme intermediate of hUGDH shows catalytic assistance from Glu161 and stabili-
zation of a tetrahedral oxyanion formed in the course of the reaction. Panel B, shown is the proposed analogous stabilization of the anionic thiohemiacetal
intermediate formed in the first oxidation step.
JANUARY 13, 2012•VOLUME 287•NUMBER 3
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
FIGURE 5. Binding of UDP-Glc at the NAD؉ site. A, shown is a surface representation of hUGDH colored with the N-terminal domain (blue) and the C-terminal
domain (red). UDP-Glc ligands from subunits A (white, C-atoms) and B (gray, C-atoms) are superimposed and shown within the NADϩ binding cleft. B, shown
is a comparison of UDP-Glc bound to E161Q (white, C-atoms) and NADH bound to wild-type hUGDH (green C-atoms, from PDB structure 2q3e). Residues within
4 Å of UDP-Glc are shown with hydrogen bonds indicated by dashed lines. Glucosyl carbon atoms discussed in the text are labeled and a water molecule that
mediates interactions between the C4 hydroxyl, and the in conformation of Gln161 is shown as a red sphere.
placement of the NADH formed in each oxidation step4 as phates, but also make an additional interaction with the side
chain of Ser130. Fig. 5B further shows that the hydroxyls at glu-
cosyl C3 and C6 of the UDP-Glc inhibitor are likewise involved
in hydrogen bonding interactions, and these were previously
well as catalytic deacylation of the enzyme. Of note, a simi-
larly flexible and functional Glu is found in aldehyde dehy-
drogenases, which resemble UGDH mechanistically in the
use of covalent catalysis from a cysteine nucleophile for the
oxidation of their aldehyde substrates (32, 34).
ϩ
not utilized for binding of NAD .
The glucosyl moiety of bound UDP-Glc is shifted ϳ5 Å com-
pared with the nicotinamide ring in the wild-type enzyme
structure, which places the glucose near the Tyr14 side chain
(supplemental Fig. S5). This shift allows Gln161 to be in the “in”
conformation without sterically interfering with binding of the
inhibitor UDP-Glc molecule. A water-mediated contact
between Gln161 and the sugar C4 hydroxyl could contribute to
stabilization of the glutamine in the in conformation. These
ϩ
Binding of UDP-Glc at NAD Binding Site; Practical Effect of
an Unexpected Observation?—In the E161Q structure, a second
UDP-Glc binds in the cleft on the surface of the N-terminal
ϩ
domain where NAD usually binds, with the glucosyl moiety
buried within the domain interface. Fig. 5 displays the molecu-
lar interactions involved in the accommodation of UDP-Glc at
ϩ
the NAD binding site. The UMP moiety of UDP-Glc is mainly
ϩ
bound through its phosphoribosyl part, which forms hydrogen
results reveal the ability of the NAD binding site of E161Q to
bonds with Asp36 and Arg41 similarly to the corresponding
exploit induced fit in the creation of an apparently fully devel-
oped binding pocket for the glucosyl moiety of a UDP-Glc
inhibitor. It could be interesting to further explore the promis-
cuity of the binding site for the purpose of targeted enzyme
inhibition.
ϩ
phosphoribosyl group of NAD in the structure of the wild-
type enzyme. Depending on the subunit, the uracil is observed
in two different conformations, with its O2 atom positioned
either toward or away from Tyr108 in the hydrophobic pocket
ϩ
that normally accommodates the adenine of NAD (Fig. 5A).
In conclusion, participation of covalent catalysis in the reac-
tion mechanism of hUGDH has been elucidated comprehen-
sively. The mechanistic proposal derived from the evidence
presented involves for the first time a clear assignment of cata-
lytic function to each individual group in the active site of the
enzyme. The UDP-Glc molecule attached to the coenzyme
binding site of thiohemiacetal enzyme intermediate of E161Q
Binding of the glucose 1-phosphate moiety of UDP-Glc
involves essentially the same residues responsible for binding of
ϩ
the nicotinamide-ribosyl-phosphate group of NAD , however,
a substantial amount of flexibility of the binding site is observed
(supplemental Fig. S5). In particular, Arg346 that originally
ϩ
interacted with the phosphate from the NAD group now
forms bidentate hydrogen bonds with the hydroxyls at glucosyl
C2 and C3 (Fig. 5B). Interestingly, a highly similar interaction
from Arg260 is utilized for accommodating the glucosyl moiety
of UDP-Glc at the substrate binding site. The UDP-Glc phos-
phates interact with the main-chain nitrogens of Tyr14 and
Val15, which are embedded within the 11GXGXXG16 repeat at
ϩ
provides the first case of NAD antagonism in hUGDH and
could, therefore, assist in the development of inhibitors of the
enzyme.
Acknowledgments—We thank Dr. Kunde Guo (Structural Genomics
Consortium) for cloning of hUGDH mutants. Dr. Panagis Filippako-
poulos, Dr. Ivan Alfano, and Prof. Stefan Knapp (all with the Struc-
tural Genomics Consortium) are gratefully acknowledged for assis-
tance, helpful discussions, and suggestions. We thank Dr. Frank von
Delft (Structural Genomics Consortium) for data collection at the
Diamond Light Source.
ϩ
the N terminus of helix ␣1 that normally binds the NAD phos-
4
The observed decrease in kcat/KmC resulting from replacement of Glu161 by
Gln is thus explained. The role of Glu161 during binding of UDP-Glc to free
hUGDH(kcat/KmS)isnot known. Considering the crystallographic data from
this and apreviousstudyofhUGDH(4), webelievethatthisrole isprobably
indirect.
2128 JOURNAL OF BIOLOGICAL CHEMISTRY
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Covalent Catalysis by Human UDP-glucose 6-Dehydrogenase
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