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Indian Journal of Chemistry
Vol. 50A, March-April 2011, pp. 355-362
Advances in Contemporary Research
Substrate orientation and the origin of catalytic power in xanthine oxidoreductase
Hongnan Cao, James Pauff† & Russ Hille*
Department of Biochemistry,The University of California, Riverside, CA 92521, USA
Email: [email protected]
Received 23 July 2010; accepted 25 August 2010
With the chemical course of the reaction catalyzed by the molybdenum-containing hydroxylase xanthine oxidoreductase
now relatively well-understood, efforts in the field have now turned to understanding the catalytic power of the enzyme in
the context of its structure. The present minireview is an account of recent efforts, from the authors’ laboratory and
elsewhere, towards understanding the role of active site amino acid residues in accelerating reaction rate. On the basis of
recent site-directed mutagenesis work, in conjunction with protein X-ray crystallography, it is now possible to attribute the
specific extent to which each contributes to transition state stabilization and the means by which this occurs.
Keywords: Bioinorganic chemistry, Catalysis, Reaction mechanisms, Molybdenum enzymes, Xanthine oxidoreductase
Xanthine oxidoreductase is the eponymous member
of a large family of molybdenum-containing enzymes
that catalyze the oxidative hydroxylation of aromatic
heterocycles and aldehydes. The enzyme has been
extensively studied for over a century, but it is only in
the past 10-15 years that a clear picture of how the
reaction proceeds and the structural context in which
this occurs has been achieved.
The structure of the bovine enzyme1 shows a
homodimer of 295 kDa with the four redox-active
centers of each subunit laid out in discretely folding
domains, from the N-terminus: a first [2Fe-2S]containing domain with a fold closely resembling
spinach ferredoxin; a second [2Fe-2S]-containing
domain consisting principally of α-helical rather
than β-sheet secondary structure; an FAD-containing
domain with a fold resembling that seen in vanillyl
oxidase family of flavoproteins; and a C-terminal
molybdenum-containing portion that consists of two
large, oblong domains lying across one another
with the molybdenum center at the domain-domain
interface. The dimer interface is in this last portion of
the polypeptide. The X-ray structure of the xanthine
dehydrogenase from Rhodobacter capsulatus has also
been determined2, and found to be very similar in
overall protein architecture to the bovine enzyme, and
particularly so with regard to the conserved amino
Present affiliation:
College of Medicine, The Ohio State University, Columbus,
OH 43210, USA.
acid residues of the active site. One important
difference is that the R. capsulatus protein is an
α2β2 heterotetramer rather than an α2 dimer like
the bovine enzyme, with the molybdenum center
in a separate subunit from the Fe/S- and flavincontaining parts of the protein. This notwithstanding,
the corresponding portions of the two proteins have
a very high degree of structural homology.
The molybdenum center of oxidized xanthine
oxidoreductase is best represented as LMoVIOS(OH),
where L is a pyranopterin cofactor coordinated
to the metal via an enedithiolate side chain that
is common to all mononuclear molybdenum- and
tungsten-containing enzymes3,4. The coordination
geometry of the molybdenum center is that of
a distorted square pyramid. Although the initial
X-ray crystal structures of xanthine oxidoreductase1
and the related aldehyde oxidoreductase from
Desulfovibrio gigas5 placed the sulfido group in
the apical position, subsequent magnetic circular
dichroism studies of xanthine oxidoreductase6
in conjunction with a comparison with subsequently
determined crystal structures of other enzymes
of the xanthine oxidoreductase family7,8, have
unequivocally demonstrated that it is the oxo
rather than sulfido group that occupies the
apical position. Furthermore, X-ray absorption
spectroscopic studies have shown that the equatorial
oxygen is present as a singly protonated hydroxide
rather than the doubly protonated water that was
originally proposed9.
Fig. 1—The reaction mechanism of xanthine oxidase. The reaction is initiated by proton abstraction from the Mo-OH group by Glu 1261,
followed by nucleophilic attack on the carbon to be hydroxylated and hydride transfer to the Mo=S. This leads to the LMoIVO(SH)(OR)
intermediate described in the text, which breaks down by electron transfer out of the molybdenum center and hydroxide displacement of
product from the molybdenum coordination sphere. The precise sequence of these last two events depends on the reaction conditions and
substrate used. The figure was generated using PyMol (PDB accession no. 1FIQ).
The reaction mechanism of xanthine oxidase as
currently understood is shown in Fig. 1. It has been
known for some time that while water is the ultimate
source of the oxygen atom incorporated into the
hydroxyl group of product in the course of the
reaction10, there is a catalytically labile site on the
enzyme that represents the proximal donor of the
catalytically introduced hydroxyl group11. This
catalytically labile oxygen has been shown to be the
equatorial Mo-OH rather than the apical Mo=O12, a
suggestion that had been made previously on the basis
of a comparison of the properties of a series of
inorganic model compounds to the enzyme active
site13. The pH dependence of the reaction of xanthine
oxidoreductase is bell-shaped, reflecting ionizations
with pKas of 6.6 and 7.4; the former has been
attributed to an active site base, and the latter to
substrate itself (reflecting the fact that enzyme works
on the neutral rather than monoionic form of
substrate)14. By analogy to the reaction mechanism
proposed for the D. gigas aldehyde oxidoreductase, in
which it was proposed that a strictly conserved active
site glutamate residue functioned as a general base in
catalysis15, the reaction of xanthine oxidoreductase is
thought to proceed via proton abstraction of the
equatorial Mo-OH by the catalytic glutamate
residue3,12, with nucleophilic attack of Mo-O- on
the carbon to be hydroxylated and concomitant
hydride transfer to the Mo=S group to give an initial
LMo O(SH)(OR), where OR represents nowhydroxylated product (uric acid in the case of
xanthine as substrate) coordinated to the molybdenum
via the catalytically introduced hydroxyl group. It is
noteworthy that earlier work with a homologous
series of substituted quinazolines had in fact
suggested that the reaction mechanism involved
nucleophilic attack on substrate16.
The LMoIVO(SH)(OR) intermediate breaks down
by displacement of product from the molybdenum
coordination sphere by hydroxide from solvent, with
electron transfer from the molybdenum to the other
redox-active centers of the enzyme accompanied by
deprotonation of the Mo-SH to return to the Mo=S of
oxidized enzyme. The sequence in which these
events occur depends on the reaction conditions and
the substrate utilized; when partial reoxidation
precedes displacement of substrate, an LMoVOS(OR)
intermediate forms that gives rise to the so-called
“very rapid” EPR signal that has long been
recognized as being mechanistically important17,18.
Under most conditions, however, product dissociation
is rate-limiting, with the rate of the chemical step
of the reaction, leading to the LMoIVO(SH)(OR)
intermediate, faster by a factor of approximately
75 (ref. 19). It is also important to recognize that
formation of this species is an oxidative event from
the standpoint of the molybdenum center, and that
the initial intermediate is formally a MoIV species20.
Although it has been suggested from time to time
that the MoV species might be formed directly by
direct one-electron transfer from substrate, the lack of
correlation between reduction potential and reaction
rate in a homologous series of substituted purines
indicates that this is not likely to be the case21.
With the reaction mechanism thus defined, the
question that remains is the origin of the catalytic
power of the enzyme, that is, the specific means
by which the transition state is stabilized in order
to accelerate reaction rate. What follows here is
first a description of the active site of the enzyme,
followed by a consideration of several protein
structures with substrate or substrate analogs
bound in the active site that, with complementary
site-directed mutagenesis studies, provide clues
as to the specific roles of amino acid residues in
transition state stabilization.
Structure of the active site
The structure of the active site of xanthine
oxidoreductase1, showing the several amino acid
residues implicated in catalysis, is given in Fig. 2.
The substrate binding site is defined by Phe 914 and
Phe 1009 (interacting with substrate in a side-on
and face-on fashion, respectively), which together
constrain the substrate to a well-defined plane relative
to the molybdenum center. There are surprisingly
few interactions between the polypeptide and the
first shell of the molybdenum coordination sphere,
with Gln 767 within hydrogen-bonding distance of
the apical Mo=O (3.12 Å nitrogen to oxygen) and
Glu 1261, the putative active site base, which lies
within hydrogen-bonding distance of the equatorial
Mo-OH of the molybdenum coordination sphere
(3.05 Å oxygen to oxygen). Gln 767 is conserved
among all eukaryotic members of the xanthine
oxidoreductase family of molybdenum enzymes, but
is not strictly conserved in the bacterial aldehydeoxidizing enzymes.
Somewhat further removed from the molybdenum
center is Glu 802 (with its nearer oxygen 3.6 Å from
the apical Mo=O and 4.1 Å from the equatorial
Mo-OH oxygen) and Arg 880 (with the nearer
guanidinium nitrogen 7.6 Å from the Mo-OH
oxygen). Both residues are universally conserved
among xanthine-oxidizing enzymes, but not among
the closely related ones utilizing aldehyde substrates.
Site-directed mutagenesis studies
Two heterologous expression systems for
functional xanthine oxidoreductase have been
developed, that of the rat enzyme in a baculovirus
system by Nishino and coworkers22, and the xanthine
dehydrogenase from R. capsulatus in E. coli by
Leimkühler and coworkers23. (A second expression
system in E. coli for the human protein yields soluble
protein, but of very low specific activity (<5%) owing
to ineffective maturation of the molybdenum center24)
Using the rat protein from the baculovirus system, it
is possible to make assignments for the two [2Fe-2S]
clusters in the enzyme, it having long been known
that two distinct EPR signals arise from the enzyme25.
Fe/S I which is readily seen at temperatures as
warm as 150 K has relatively sharp features and
g1,2,3 = 2.022, 1.932 that are typical of [2Fe-2S]
clusters. By contrast, Fe/S II has much broader
features and g1,2,3 = 2.110, 1.991 and 1.894 (ref. 26)
Fig. 2—Structure of the active site of bovine xanthine oxidase.
Specific amino acid residues involved in substrate binding and
catalysis, as described in the text, are shown. The figure was
generated using PyMol (PDB accession no. 1FIQ).
and is only observed below 50 K. It has been known
for some time that the reduced Fe/S I interacts
magnetically with the molybdenum center when the
latter is in the Mo(V) oxidation state27, but in the
crystal structure of the protein it is the cluster in
the unusual α-helical domain rather than the more
normal ferredoxin-like domain that lies closer to
the molybdenum center. Using the rat xanthine
oxidoreductase from the baculovirus system, Nishino
and coworkers28 were able to demonstrate that
mutation of cysteine residues in the ferredoxin-like
domain perturbed the Fe/S II EPR signal, while
similar mutations in the α-helical domain perturbed
the Fe/S I signal. Thus, counterintuitively, the
[2Fe-2S] cluster with the more normal EPR
characteristics was shown to reside in the more
unusual polypeptide fold.
Mutagenesis of amino acid residues in the active
site have been largely restricted to work with the
R. capsulatus system, where adequate amounts of the
recombinant proteins are available for detailed kinetic
characterization. Leimkühler et al.29 demonstrated
that mutation of Glu 232 (equivalent to Glu 802 in the
bovine enzyme) to Ala reduced kcat in steady-state
experiments from 108 s-1 to 4.4 s-1, and kred in the
anaerobic reduction of enzyme by xanthine from
67 to 5.5 s-1. Very similar results were seen in the
steady-state kinetics of an E803V mutant of the
E. coli-expressed human xanthine oxidoreductase24.
In the rapid reaction work with the R. capsulatus
enzyme, the affinity of enzyme for substrate also
decreased by a factor of approximately 10, with
Kd increasing from 34 to 409 µM; kred/Kd, reflecting
the second-order reaction of free enzyme with
free substrate through the first irreversible step of
the reaction (in this case, through to formation of
the LMoIVO(SH)(OR) intermediate) decreased by
two orders of magnitude from 1.97 × 106 M-1s-1 to
1.34 × 104 M-1s-1. At face value, the effect on kred and
Kd is such that the interaction between Glu 232 and
substrate provides some 3 kcal/mol of free energy,
approximately half of which is utilized to bind
substrate (as reflected in the higher Kd seen in the
mutant) and half to stabilize the transition state
(as reflected in the lower kred exhibited by the mutant).
Given that the chemical step of the reaction is likely
to become rate-limiting in the mutant, however, and
assuming that with wild-type enzyme it is not
(and to the same degree as is seen with the vertebrate
enzymes, i.e., by a factor of 75), it is likely that the
actual free energy available from the interaction
is ~5.5 kcal/mol, of which 4 kcal/mol is utilized to
stabilize the transition state for the chemical step of
the reaction rather than simply bind the substrate. On
the basis of a computational study of the relative
stabilities of several tautomers of free xanthine
and the LMoIVO(SH)(OR) intermediate, it has been
suggested that Glu 232 accelerates reaction rate
specifically by facilitating a tautomerization in the
course of nucleophilic attack that involves proton
transfer from N3 to N9 of the purine ring and serves to
compensate for the negative charge accumulating on
the imidazole subnucleus of the purine in the course
of the reaction30. This is illustrated in Fig. 3.
Glu 197 (Glu 767 in the bovine enzyme) was also
mutated to an alanine. This residue hydrogen-bonds
to the apical Mo-O of the molybdenum coordination
sphere and does not constitute part of the substrate
binding site. Consistent with this, in rapid reaction
studies Kd for substrate was unchanged, while the
limiting rate constant for reduction at high [xanthine]
was reduced by a factor of seven from that seen with
wild-type enzyme (15 s-1 versus 108 s-1, respectively).
Again, given the likelihood that there has been a
Fig. 3—The proposed role of R. capsulatus xanthine
dehydrogenase Glu 232 (Glu 802 in the bovine enzyme) in
facilitating tautomerization in the course of the reaction.
change in rate-limiting step in the overall reaction,
from product dissociation to the initial formation
of the LMoIVO(SH)(OR) intermediate as discussed
above, the actual effect of the mutation on the
rate constant for the first step of the reaction may
be as great as 500-fold, implying that the Glu 197
hydrogen bond to the Mo=O contributes several
kcal/mol to transition state stabilization, presumably
by modulating the electronic structure (and hence
reactivity) of the molybdenum center.
A series of mutants at Glu 730, the putative active
site base equivalent to Glu 1261 in the bovine
enzyme, were also examined in this study. In no case
was discernible activity detected in steady-state
assays, nor was any detectable reduction observed in
anaerobic reductive half-reaction experiments. Even
taking into account the variable amount of functional
molybdenum center in the mutants (as low as 33 %
in the case of the E730A mutant, as compared with
76 % in the wild-type enzyme), the loss of activity
was profound. Whereas 100 µM xanthine reduces
the wild-type enzyme to completion in ~100 ms, the
E730A was not perceptibly reduced in an overnight
incubation; conservatively, this amounts to at least
a 107-fold reduction in the limiting rate of enzyme
reduction. Clearly, Glu 730 is extremely important
catalytically, contributing at least 10 kcal/mol in
transition state stabilization. That Glu 730 is working
specifically as an active site base is suggested by
the fact that the E730A mutant is partially active at
pH 10, where the Mo-OH is expected to spontaneously
deprotonate (Ibdah & Hille, unpublished data).
The final active site residue examined has been is
Arg 310 (Arg 880 in the bovine enzyme), which has
been mutated to methionine31. This residue lies over
7 Å from the catalytically labile Mo-OH and was
expected to be involved primarily in substrate binding
rather than transition state stabilization. Surprisingly,
the principal effect was a 4000-fold reduction in kred,
indicating that Arg 310 contributed significantly to
transition state stabilization, by at least 4.5 kcal/mol.
Consistent with these results, a homologous R881M
mutant in the human enzyme lacked activity in
steady-state assays24. Interestingly, in a comparison
of a homologous series of purine substrates, all
hydroxylated at the 8 position as is xanthine, it was
found that the substrates fell into two categories: a
first, including xanthine, that were effective substrates
for wild-type enzyme and which were profoundly
affected by the R310M mutation, and a second
consisting of poor substrates for wild-type enzyme but
which were minimally affected by the mutation31.
Fig. 4—The proposed role of R. capsulatus xanthine dehydrogenase Arg 310 (Arg 880 in the bovine enzyme) in transition state
stabilization in the course of nucleophilic attack.
Fig. 5—Proposed good and bad substrate orientations in the active site of xanthine oxidase31.
On the basis of these results, it has been suggested
that Arg 310 stabilizes the transition state for the
chemical step of the reaction by providing charge
compensation for the negative charge accumulating
on the heterocycle, as illustrated in Fig. 4.
Structural studies of substrate binding
The above proposals for the catalytic roles of
Glu 232/802 and Arg 310/880 depend critically on
substrate being positioned in the active site as shown
in Figs 3 and 4, with confined to the plane defined by
the two phenylalanine residues of the active site.
An upside down orientation such as shown in Fig. 5,
prevents both residues from functioning effectively,
and is the proposed structural basis for the lower
activity exhibited by some purines relative to others31.
The question is whether this is in fact the case. The
first crystallographic evidence as to the orientation
of substrate in the active site came from the
structure of the R. capsulatus enzyme in the complex
with the inhibitor alloxanthine2. Alloxanthine, a
pyrazolopyrimidine analog of the purine xanthine,
is a mechanism-based inhibitor of xanthine
oxidoreductase that has long been known to form a
very stable complex with the reduced form of the
enzyme32. In the crystal structure, alloxanthine is
oriented in the active site as shown in Fig. 6. There is
a direct bond between N-2 of the pyrazole subnucleus
of the inhibitor and the molybdenum (displacing the
Fig. 6—The structure of reduced R. capsulatus xanthine
dehydrogenase in complex with the inhibitor alloxanthine.
Specific amino acid residues are shown. The figure was generated
using PyMol (PDB accession no. 1JRP).
catalytically labile Mo-OH from the molybdenum
coordination sphere), and its pyrimidine subnucleus is
oriented with the “distal” C-6 keto group (equivalent
to the C-2 keto group of xanthine, owing to
the different numbering conventions for the two
heterocycles) pointing toward Arg 310. Interestingly,
in a complex of alloxanthine with the oxidized rather
than reduced (bovine) enzyme, inhibitor is bound in
the opposite orientation (Fig. 5), with the “proximal”
C-4 carbonyl (equivalent to C-6 of purine substrates)
interacting with Arg 880 (Nishino T & Pai E, personal
communication). In this structure, as expected,
there is no bond between the N-2 nitrogen and
molybdenum. On the basis of the above discussion of
purine substrates, this second orientation represents
the catalytically effective one; in the first (with
reduced enzyme), as a result of the closer position
required for direct coordination of inhibitor to the
molybdenum, the heterocycle binds in an inverted
orientation, with the “distal” rather than “proximal”
keto group of the pyrimidine subnucleus interacting
with the active site arginine.
In order to explicitly examine substrate orientation
in the active site and test the validity of the proposed
catalytic roles of the various active site residues,
we have examined the X-ray crystal structures of
the bovine enzyme in complex with a variety of
substrates and substrate analogs. The first to
be examined was 2-hydroxy-6-methylpurine, a
slow substrate for the enzyme20. Using this poor
substrate, the structure shown in Fig. 7A is observed33
(at a resolution of 2.3 Å), in which bridging electron
density between the molybdenum and C-8 of the
heterocycle is clearly seen. This can only mean
that catalysis has been initiated in the crystal, with
the observed structure that of the LMoIVO(SH)(OR)
intermediate or its LMoVOS(OR) oxidation product
(probably the latter since the observed Mo-S distance
of 2.0 ± 0.2 Å suggests a Mo=S rather than MO-SH
ligand). The orientation of substrate, and in particular
the Mo-C distance of 3.4 Å, is generally consistent
with ENDOR studies of the “very rapid” species
Fig. 7—The X-ray crystal structures of xanthine oxidase in complex with various substrates. [(a) functional enzyme and 2-hydroxy-6methylpurine33, (PDB 3BNJ); (b) nonfunctional, desulfo enzyme and xanthine35, (PDB 3EUB); (c) functional enzyme and lumazine35
(PDB 3ETR); (d) functional enzyme and hypoxanthine in two different orientations of substrate36 (PDB 3NRZ). All figures were
generated using COOT].
which indicated that this interatomic distance was too
large for a direct Mo-C bond and that the product
was coordinated to the metal in a simple end-on
manner34, as is seen crystallographically.
In this structure, the asymmetric electron density
seen for the substrate in the active site clearly
indicates that the orientation of the coordinated
product (2,8-dihydroxypurine) in the active site is
such that the “distal” C-2 keto group of the
pyrimidine subnucleus is oriented toward Arg880, and
is within hydrogen-bonding distance of ~ 3.1 Å.
2-Hydroxy-6-methylpurine is a poor substrate for
xanthine oxidase and one that is only modestly
affected by the R330M mutation in the R. capsulatus
enzyme, the orientation seen here is presumably that
of the less effective catalytic orientation, and is in
agreement with that expected on the basis of the
argument above.
The next structures that have examined are those of
the nonfunctional desulfo form of the enzyme in
complex with xanthine (at 2.6 Å resolution, Fig. 7B),
and of the functional form in reaction with the pteridine
substrate lumazine (at 2.2 Å resolution, Fig. 7C)35.
Both these substrates are “good” substrates in the
context of the orientation discussion. In both
structures, the orientation of the pyrimidine
subnucleus of the heterocycle is found to be opposite
to that seen with the poor substrate, 2-hydroxy-6methylpurine, with the “proximal” C-6 rather than
C-2 oriented toward the active site arginine, 2.9 Å
away in the case of the xanthine structure, and 3.5 Å
in the case of the lumazine structure. This orientation
is that expected to be catalytically more effective on
the basis of the argument above, and the prediction
that good substrates bind in a different orientation
than poor substrates is borne out by the observed
crystal structures. Both these structures have
subsequently been resolved to better than 1.8 Å
resolution. On the basis of the published crystal
structures to date, it is evident that purine substrates
that are hydroxylated at the 8 position do indeed bind
to xanthine oxidase in two different ways, as proposed
on the basis of the kinetic work described above. The
more catalytically effective orientation is such that
Glu 802 is indeed positioned appropriately to function
catalytically in facilitating tautomerization between
N-3 and N-9, and Arg 880 by stabilizing negative
charge accumulation on the C-6 carbonyl of substrate
in the course of nucleophilic attack.
Physiologically, xanthine oxidase functions not
only to hydroxylate xanthine at C-8 to give uric acid,
but also hypoxanthine at C-2 to give xanthine in the
immediately preceding step of purine catabolism.
Glu 802
Glu 802
Fig. 8—Proposed role of Glu 802 in facilitating the
tautomerization of hypoxanthine in the course of hydroxylation by
xanthine oxidase.
To examine the role of amino acid residues in
catalysis of C-2 of purine substrates, crystal structures
of bovine xanthine oxidase in complex with
hypoxanthine (at 1.8 Å resolution) and the
chemotherapeutic agent 6-mercaptopurine (at 2.6 Å
resolution) have been determined36. For each,
different orientations of substrate are observed in
the two active sites of the dimeric enzyme (there
being one dimmer per asymmetric unit in the crystal).
One orientation is appropriate for hydroxylation at
C-2 of substrate, leading to xanthine or 6-thioxanthine
as product (Fig. 7D). The other has C-8 oriented
toward the active site molybdenum center (Fig. 7E),
as if to give 6,8-dihydroxypurine, a putative
product not previously thought to be formed by the
enzyme. Separate kinetics studies demonstrate
that hypoxanthine is hydroxylated essentially
quantitatively at C-2, indicating that the second
substantially less catalytically effective than the
former, so that even when hypoxanthine binds in
an orientation that permits hydroxylation at C-8
the reaction proceeds only very slowly36. The
apparent enzyme selectivity for C-2 over C-8 with
hypoxanthine appears to be due to both relatively
subtle differences in substrate orientation in the
active site and to differences in the intrinsic
reactivity of the two sites; a computational analysis
of hypoxanthine indicates that the C-2 carbon is
significantly more positively charged than C-8,
making it intrinsically more susceptible to
nucleophilic attack. Interestingly, these studies
show that, in contrast to the situation with xanthine,
the preferred tautomer of hypoxanthine with N-9 of
the imidazole subnucleus is (modestly) more stable
than that with N-7 protonated36. This being the
case, Glu 802 is again positioned to facilitate
tautomerization in the course of nucleophilic attack,
this time from N-9 to N-3 (the reverse sense
as proposed in the hydroxylation of xanthine at C-8),
as illustrated in Fig. 8.
A combination of site-directed mutagenesis work
and crystallographic studies has substantiated the
reaction mechanism of xanthine oxidase as it is
presently understood and has provided insight into
the specific catalytic roles of active site residues.
The majority of the catalytic power of the enzyme
can be attributed to the action of (1) Glu 1261
(Glu 730 in the R. capsulatus enzyme) as an active
site base, deprotonating the Mo-OH group of
the molybdenum coordination sphere and thereby
facilitating nucleophilic attack on the carbon center
to be hydroxylated; (2) Glu 802 (232) in facilitating
tautomerization of substrate, thereby stabilizing
negative charge accumulation on the heterocycle
in the course of nucleophilic attack; and (3) Arg 880
(310) in formal charge compensation in the course
of nucleophilic attack on substrate. Together, these
three residues are responsible for at least 17 kcal/mol
in transition state stabilization (equivalent to twelve
orders of magnitude in rate acceleration).
This work was supported by a grant from the US
National Institutes of Health (GM 075036 to RH).
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