Báo cáo khoa học: Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190 pot

Probing the catalytic potential of the hamster arylamine
N-acetyltransferase 2 catalytic triad by site-directed
mutagenesis of the proximal conserved residue, Tyr190
Xin Zhou
1
, Naixia Zhang
2
,LiLiu
1
, Kylie J. Walters
2
, Patrick E. Hanna
1
and Carston R. Wagner
1
1 Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA
2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
Introduction
Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) are
ubiquitous enzymes in nature that catalyze the N-acety-
lation of arylamines and the O-acetylation of arylhydr-
oxylamines, as well as the N,O-transacetylation of
arylhydroxamic acids [1]. These reactions result in the
detoxification of arylamine and arylhydrazine drugs,
such as isoniazid, sulfonamides, procainamide and
hydralazine, reducing the potential for cytochrome
P450-dependent N-oxidation [2,3], which is also respon-
sible for the bioactivation of arylamine environmental
toxicants, such as 2-aminofluorene, 4-aminobiphenyl
and 2-amino-1-methyl-6-phenylimidazo(4,5-b)-pyridine
[4,5]. Humans express two NAT isozymes (NAT1 and

NAT2), which have 81% sequence identity, but differ
Keywords
arylamine; carcinogen; N-acetyltransferase;
NAT; kinetics; pK
a
Correspondence
C. R. Wagner, Department of Medicinal
Chemistry, University of Minnesota, 8-174
Weaver Densford Hall, 308 Harvard St. S.E.,
Minneapolis, MN 55455, USA
Fax: +1 612 624 0139
Tel: +1 612 624 2614
E-mail:
(Received 14 July 2009, revised 3
September 2009, accepted 17
September 2009)
doi:10.1111/j.1742-4658.2009.07389.x
Arylamine N-acetyltransferases (NATs) play an important role in both the
detoxification of arylamine and hydrazine drugs and the activation of aryl-
amine carcinogens. Because the catalytic triad, Cys-His-Asp, of mammalian
NATs has been shown to be essential for maintaining protein stability, ren-
dering it impossible to assess alterations of the triad on catalysis, we
explored the impact of the highly conserved proximal residue, Tyr190,
which forms a direct hydrogen bond interaction with one of the triad resi-
dues, Asp122, as well as a potential pi-pi stacking interaction with the
active site His107. The replacement of hamster NAT2 Tyr190 by either
Phe, Ile or Ala was well tolerated and did not result in significant altera-
tions in the overall fold of the protein. Nevertheless, stopped-flow and
steady-state kinetic analysis revealed that Tyr190 was critical for maximiz-
ing the acetylation rate of NAT2 and the transacetylation rate of p-amino-

benzoic acid when compared with the wild-type. Tyr190 was also shown to
play an important role in determining the pK
a
of the active site Cys during
acetylation, as well as the pH versus the rate profile for transacetylation.
We hypothesized that the pH dependence was associated with global
changes in the active site structure, which was revealed by the superposi-
tion of [
1
H,
15
N] heteronuclear single quantum coherence spectra for the
wild-type and Y190A. These results suggest that NAT2 catalytic efficiency
is partially governed by the ability of Tyr190 to mediate the collective
impact of multiple side chains on the electrostatic potential and local con-
formation of the active site.
Abbreviations
AcCoA, acetyl-coenzyme A; HSQC, heteronuclear single quantum coherence; NAT, arylamine N-acetyltransferase; PABA, p-aminobenzoic
acid; pABglu, p-aminobenzoyl-glutamic acid; PNA, p-nitroaniline; PNP, p-nitrophenol; PNPA, p-nitrophenyl acetate.
6928 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
in substrate specificity and tissue distribution [6–8].
Human NAT2, which is found mainly in the liver [9]
and the intestine [10], selectively acetylates substrates
such as isoniazid, sulfamethazine, daspone and procain-
amide [11], whereas human NAT1, which is extensively
distributed and expressed early in development at the
blastocyst stage [8], preferentially acetylates substrates
such as p-aminobenzoic acid (PABA), p-aminosalicylic
acid and p-aminobenzoyl-glutamic acid (pABglu)
[6,12]. The widespread expression of human NAT1 and

the selectivity for pABglu, as well as the presence in
blastocytes and fetal tissues of NAT1, has suggested
that this enzyme may have a role in folate metabolism
and neural tube development [13,14].
Because many human NAT substrates are carcino-
gens and drugs, elucidation of the catalytic mechanism
of these enzymes would allow a more comprehensive
understanding of the origin of substrate specificity and
structure ⁄ function relationships. Previously, studies of
initial velocity patterns and product inhibition of NAT
from rabbit, pigeon, Mycobacterium tuberculosis and
Pseudomonas aeruginosa suggested a Ping Pong Bi Bi
mechanism involving the formation of an acetylated
enzyme intermediate [15–20]. The acetylated cysteinyl
enzyme intermediate was isolated after incubation
of rabbit liver NAT with [2-
3
H] acetyl-coenzyme A
(AcCoA) in the absence of amine [21] and the active
site Cys68 or Cys69 has been further identified through
thiol-specific modification and site-directed mutagene-
sis [22–24]. The first crystal structure of NAT, from
Salmonella typhimurium (PDB code: 1E2T), revealed a
strictly conserved Cys-His-Asp catalytic triad, reminis-
cent of Cys proteases [14]. Site-directed mutagenesis
experiments with NATs have confirmed that each resi-
due of the triad is individually essential for catalysis
and protein stability [24–26].
Our laboratory has investigated the individual steps
of the catalytic mechanism of hamster NAT2 [26,27],

which shares > 60% sequence identity and similar
substrate specificity with human NAT1 [28–31]. The
catalytic mechanism for hamster NAT2, and by anal-
ogy all NATs, proceeds through rapid formation of an
acyl-Cys intermediate, followed by rate-limiting acyl
transfer [27]. The exceptional reactivity of the active
site Cys68 can be attributed to the formation of a thio-
late–imidazolium ion pair with a pK
a
of 5.2 [26]. How-
ever, in contrast to Cys proteases, which typically
exhibit an additional basic limb p K
a
of 8–9, the second
pK
a
for hamster NAT2 acetylation was found to be
> 9.5. For both NATs and Cys proteases, the basic
pK
a
has been attributed to the triad His [26,32].
Elucidation of the influence of His107 and Asp122 on
the catalytic reactivity of Cys68 has remained elusive, as
mutations at these two positions (e.g. D122N, D122A,
H107Q, H107N) generate insoluble protein with no
detectable activity, even after refolding [26,27]. Conse-
quently, we hypothesized that modulation of the cata-
lytic potential of the catalytic triad might be accessible
through point site mutations of the proximal residue,
Tyr190, which is highly conserved and participates in

hydrogen bonding with Asp122 [2.6 A
˚
in S. typhimurium
NAT crystal structure (PDB code: 1E2T) [14], 2.8 A
˚
in
human NAT1 crystal structure (PDB code: 2QPT) [33]
and 3.28 A
˚
in hamster NAT2 model structure, as well as
potential P-P interactions with His107 (Fig. 1). This
Tyr is highly conserved [34–36] in all the NAT sequences
reported to date, with the only exception being the iso-
form banatB from Bacillus anthracis, where a His is at
the equivalent position [36]. In addition, there is an array
of known NAT polymorphisms, some of which have
been associated with an increased cancer risk [37]. Gen-
erally, these mutations have resulted in a loss of NAT
activity due to either catalytic triad mutations, decreased
enzyme stability or sequence truncation [3]. We hypothe-
A
B
Fig. 1. Model structure of hamster NAT2 demonstrates that the
residue Y190 is proximal to the catalytic triad. (A) Ribbon represen-
tation of the model structure of hamster NAT2. The catalytic triad
is colored in red and Y190 is in green. (B) Expanded view of
selected residues of hamster NAT2. Residues are colored by atom
type: carbon, nitrogen, sulfur and oxygen in white, blue, orange and
red, respectively.
X. Zhou et al. The catalytic role of Y190 in NAT

FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6929
sized that unlike other active site residues, mutations at
Tyr190 might be tolerated, despite its conservation, as
inactive NAT polymorphisms at this position have not
been identified [3]. Furthermore, active genetic variants
at position 190 have been identified by chemical muta-
genesis [38].
Consequently, we carried out steady-state and tran-
sient-state kinetic studies on a series of mutants at this
position to delineate the contribution of the hydroxyl
moiety (Tyr190 to Phe), aromatic stacking (Tyr190 to
Ile), and interior side chain packing (Tyr190 to Ala)
on the catalytic and structural integrity of the enzyme.
In addition, the impact of the most disruptive mutant,
Tyr to Ala, at this position on the active site structure
was characterized by NMR spectroscopy.
Results
CD spectroscopy and HSQC analyses of
15
N-labeled Y190A and wild-type NAT2
Similar CD spectra were observed for wild-type and
Y190 mutants at pH 7 (see Fig. S1), which further
confirmed that the Y190 mutations, unlike the H107
and D122 mutations, did not disrupt the overall sec-
ondary structure composition of the protein [26,27].
To probe the structural implications of Y190 muta-
tions more deeply, we used [
1
H,
15

N] heteronuclear
single quantum coherence (HSQC) experiments to
record the chemical shift values of NAT amide nitro-
gen and hydrogen atoms.
15
N-labeled proteins were
prepared and [
1
H,
15
N] HSQC experiments carried out;
the resulting spectra collected at 600 MHz were super-
imposed. Consistent with the CD spectra, the amide
resonances of most of the residues in secondary struc-
tural elements were unperturbed; however, the Y190A
mutation caused nearly all of the amides of residues in
the catalytic cavity to shift (Fig. S2A). Such shifting is
caused by changes in the atom’s chemical environment,
and the affected residues included those proximal to
Y190, such as H107, D122, F125 and F192, the latter
of which forms an edge-to-face aromatic stacking with
Y190 (Fig. S2B). However, also included were L69,
S224 and F288, which are up to 18A
˚
away from
Y190’s side chain. Although the amide resonance for
C68 was not observable, the amide resonances of
H107 and D122 were shifted, indicating that mutation
of Tyr190 disturbs the conformation of these catalytic
triad residues [35]. In addition, residues close to D122

(I120, V121, A123 and G124), and residue L69, close
to C68, and residue L108, close to H107, were
affected. Changed chemical shifts of F125 and F192,
which form the edge-to-face aromatic stacking with
Y190, were also observed (Fig. S2B). In addition to
the observed shifting, residue attenuation was
observed, most obviously for L69, L108, D122 and
A123. Such attenuation is caused by chemical
exchange and suggests that the catalytic cavity configu-
ration compensates for the loss of Y190.
Comparison of specific activities with
p-nitrophenyl acetate (PNPA)
⁄
PABA
Because the Y190 mutants are correctly folded, as
shown by CD, the specific activity was determined as
the transacetylation reaction rate of PNPA ⁄ PABA
catalyzed by wild-type and Y190 mutants with saturat-
ing PNPA concentrations and fixed PABA and NAT2
enzyme concentrations. The measured activities were
184 ± 8, 130 ± 21, 22 ± 3 and 8.5 ± 0.7 lmolÆmg
)1
Æ-
min
)1
for wild-type, Y190F, Y190I and Y190A, respec-
tively. Therefore, under the given conditions,
eliminating the hydroxyl group of Tyr190 by the
mutation Y190F in NAT2 yielded only a modest
decrease of 30% in activity relative to the wild-type

enzyme. However, the Y190I and Y190A mutations had
substantial effects, resulting in losses of activity of 88%
and 95%, respectively, relative to wild-type enzyme.
Presteady-state kinetics of NAT acetylation
The rate of acetylation of NATs was determined with a
stopped-flow apparatus by measuring the fast release of
p-nitrophenol (PNP) before the acetylated enzyme con-
centration reaches steady state. Each of the Y190
mutants demonstrated similar ‘burst kinetics’ as
observed for the wild-type [26], indicating the formation
of the acetylated enzyme intermediate. Overall, the
second-order rate constant, k
2
⁄ K
m
acetyl
, for the Y190
mutants was 2–20-fold lower than the value observed
for the wild-type ( Table 1), indicating a slower rate of
enzyme acetylation. The decrease in the k
2
⁄ K
m
acetyl
value was largely due to a decrease in k
2
, rather than a
significant change in K
m
acetyl

. In the case of the Y190F
mutant, the value of k
2
decreased slightly from
1301 ± 716 s
)1
(wild-type) to 279 ± 54 s
)1
; however, a
pronounced k
2
decrease was observed for both the
Y190I mutant (57 ± 6 s
)1
) and the Y190A mutant
(15 ± 3 s
)1
) of nearly 23- and 87-fold, respectively.
Consequently, Y190 appears to be necessary for main-
taining the optimal reactivity of Cys68 for acetylation.
Steady-state kinetics of acetyl-enzyme hydrolysis
As previously demonstrated by single turnover kinetics
with PNPA, acetylation of wild-type NAT proceeds
The catalytic role of Y190 in NAT X. Zhou et al.
6930 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
through rapid formation of an enzyme intermediate
(k
2
) followed by rate-limiting hydrolysis (k
hydrolysis

) [26]
(Scheme 1). Each of the Y190 mutants exhibited simi-
lar burst kinetics, followed by rate-limiting deacetyla-
tion by water (Table 1). Nevertheless, the rate of
hydrolysis (k
hydrolysis
) for each mutant was found to
have significantly increased by 4–30-fold, relative to
the wild-type, resulting in a 3.5–40-fold decrease in the
lifetime of the acetylated enzyme. Removal of the
para-hydroxyl group by the Tyr190 to Phe mutation
resulted in a decrease in the rate of enzyme acetylation
(k
2
) by 4.7-fold and an increase in the rate of interme-
diate hydrolysis (k
hydrolysis
) of 3.6-fold. Similarly, a
decrease in k
2
and an increase in k
hydrolysis
($ 29-fold)
was observed when the phenol moiety of Tyr190 was
replaced by the sec-butyl group of Ile. The Tyr190 to
Ile mutation resulted in the largest decrease in acety-
lated enzyme stability. When the Tyr190 side chain
was deleted entirely, a reduction of nearly 90-fold in k
2
was observed. However, the value of k

hydrolysis
was
only increased by 4.7-fold. Thus, although a reduction
in hydrogen bonding ability and replacement of the
aromatic ring with an aliphatic side chain appear to
have similar, but opposite, impacts on NAT acetyla-
tion and deacetylation, perturbation of the active site
by complete removal of the side chain mainly affected
enzyme acetylation (k
2
).
pH dependence of NAT acetylation
Usually, the pH dependence of acetylation of NAT
(k
2
⁄ K
m
aceytl
) reflects ionizations of the free enzyme
and free substrate that are either directly or indi-
rectly involved in substrate binding and in the cata-
lytic process [39]. For the wild-type, the pH
dependence of the single turnover rate constant,
k
2
⁄ K
m
acetyl
, fit best to a model for two pK
a

values,
with the first, pK
a1
acetyl
(5.16 ± 0.14), assigned to
the active site Cys, and the second, pK
a2
acetyl
(6.79 ± 0.25), assigned to a probable conformational
change [26]. In the case of Y190F, the value of log
(k
2
⁄ K
m
acetyl
) rose as a function of pH until a plateau
was reached above pH 7.5. The data were fit into a
one-pK
a
model with a pK
a
acetyl
value of 5.16 ± 0.05,
which is virtually identical to the first pK
a1
acetyl
(5.16 ± 0.14) obtained for wild-type NAT2 (Fig. 2).
This suggests that removal of the hydroxyl group
from Y190 results in little perturbation of the active
site Cys pK

a
, which is consistent with the slightly
reduced acetylation rate k
2
. However, in contrast
with the pH profile for wild-type NAT2, where the
maximum k
2
⁄ K
m
acetyl
was reached at pH 6.4, the
k
2
⁄ K
m
acetyl
for Y190F was pH independent under
neutral and basic conditions. Therefore, the modest
reduction in the acetylation rate, as well as the lack
of the second pK
a
acetyl
, suggests that Y190 may be
important in communicating the pH-dependent con-
formational change at the active site. The more dras-
tic mutations, Y190I and Y190A, however, revealed
the importance of the phenyl ring of the Tyr side
chain on the pH dependence of enzyme acetylation.
Both of the pH profiles fit best to a two-pK

a
model
with the pK
a1
acetyl
values being elevated by one unit
(i.e. Y190I 6.24 ± 0.16, Y190A 6.00 ± 0.07, com-
pared with wild-type 5.16 ± 0.14). Thus, the reactiv-
ity of the catalytic Cys was reduced for these two
mutants, which is consistent with the significant
decrease in the observed rate of acetylation.
pH dependence of transacetylation by NAT
The pH dependence of transacetylation of PNPA ⁄
PABA (k
cat
⁄ K
PABA
) reflects the ionization of groups
on the acetylated enzyme and ⁄ or PABA that are
either directly or indirectly involved in catalysis or
binding of the substrate during the deacetylation
E + PNPA
k
1
k
–1
E PNPA
pNP
AcE + H
2

O
k
2
AcOH
k
hydrolysis
E
Scheme 1. Acetylation of NAT and hydrolysis of acetylated
enzyme intermediate.
Table 1. Presteady-state kinetics of single turnover reactions of hamster NAT2 acetylation by PNPA. k
hydrolysis
, the hydrolysis rate constant
of the acetylated enzyme intermediate; T
1 ⁄ 2
, the half-life time of the acetyl-enzyme intermediate.
Hamster NAT2
K
m
acetyl
(mM) k
2
(s
)1
)
k
2
⁄ K
m
acetyl
(M

)1
Æs
)1
)
k
hydrolysis
(s
)1
)
T
1 ⁄ 2
(s)
Wild-type
a
9 ± 5.7
a
1301 ± 716
a
(1.4 ± 0.05) · 10
5a
(7.9 ± 0.7) · 10
)3a
88 ± 8
a
Y190F 3.5 ± 1.1 279 ± 54 (8.0 ± 3.9) · 10
4
(28 ± 0.4) · 10
)3
25 ± 3
Y190I 4.1 ± 0.7 57 ± 6 (1.4 ± 0.04) · 10

4
(230 ± 40) · 10
)3
1.9 ± 0.1
Y190A 2.8 ± 0.7 15 ± 3 (5.5 ± 2.3) · 10
3
(37 ± 8) · 10
)3
19 ± 0.4
a
Values for the ‘wild-type’ protein are taken from [26].
X. Zhou et al. The catalytic role of Y190 in NAT
FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6931
step. For wild-type NAT2, the pH influence on
k
cat
⁄ K
PABA
revealed only one pK
a
transacetyl
at
5.55 ± 0.14 with two active forms and a (k
cat
⁄
K
PABA
)
lim
of 3000 ± 50 mm

)1
Æs
)1
. The ratio, r, was
calculated to be 0.13 ± 0.04, indicating that the
deprotonated form of the enzyme is about 8-fold
more active than the protonated form [27]. However,
for the three mutants, the pH profiles were best
fitted to a two-pK
a
model with two active forms,
and decreasing (k
cat
⁄ K
PABA
)
lim
values (Fig. 3). In our
previous solvent isotope effect study of wild-type
NAT2, a normal solvent kinetic isotope effect
[
H ⁄ D
(k
cat
⁄ K
b
)
lim
= 2.01 ± 0.04] across the entire pH
range for PNPA and PABA was consistent with a

general base catalysis. Previously, the active site
His107 was identified as the probable base with a
pK
a
transacetyl
of 5.55 for the acetylated enzyme [27].
We assumed general base catalysis was also
employed by the Y190 mutants, thus, the first
pK
a1
transacetyl
values from the fitting results were
assigned to His107 for the acetylated mutant NAT2.
Accordingly, the transacetylation of PNPA ⁄ PABA by
Y190F proceeded with a pK
a1
transaccetyl
of
5.48 ± 0.06, which is similar to the pK
a
transacetyl
of
the wild-type, and consistent with a transacetylation
rate similar to the wild-type. In contrast, the
pK
a1
transacetyl
values 6.56 ± 0.12 and 6.40 ± 0.12, for
Y190I and Y190A, respectively, reflect their signifi-
cantly lower transacetylation rates and, thus, the

overall importance of Y190 on the protonation state
of His107.
Kinetic parameters for transacetylation of
arylamine substrate and Brønsted plot
The transacetylation of arylamine (k
4
)(Scheme 2) from
acetylated NAT2 proceeds much faster (1000–10 000-
fold) than hydrolysis of the acetylated NAT intermedi-
ate (k
hydrolysis
) [27] (Scheme 1). Using PNPA or
AcCoA as the acetyl donor and PABA, anisidine,
pABglu or p-nitroaniline (PNA) as the acetyl acceptor,
the steady-state kinetic parameters for transacetylation
by the Y190 mutants were determined at 25 °C, pH
7.0 (Table 2). Previously we have shown that for reac-
tions with PNPA as the acetyl donor, the transacetyla-
tion of arylamine substrate (k
4
), rather than the
acetylation of NAT2 (k
2
), is the rate-limiting step [27].
Therefore, the k
cat
values for PNPA ⁄ anisidine,
PNPA ⁄ PABA, PNPA ⁄ pABglu approximate k
4
(the

rate of transacetylation of amine acceptors) (Eqn 1).
AB
CD
Fig. 2. pH dependence of hamster NAT2
single turnover by PNPA.
The catalytic role of Y190 in NAT X. Zhou et al.
6932 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
However, for reactions employing AcCoA as the acetyl
donor, the k
cat
values are determined by the individual
rate constants for both the acetylation (k
2
) and deacet-
ylation (k
4
) steps (Eqn 2) [27]. On the basis of the
Ping Pong mechanism, the acetylation rate (k
2
)of
NAT2 by the acetyl donor is independent of the trans-
acetylation rate (k
4
) of the arylamine substrate.
Because the values of k
4
for PABA can be inferred
from the PNPA ⁄ PABA reaction, which are in the
range of 38–620 s
)1

, based on the k
cat
values for
PABA acetylation by AcCoA (Eqn 2), the values of k
2
for AcCoA can be predicted to range from 10 to
1740 s
)1
. Hence, from the k
cat
values for the acetyla-
tion of PNA by AcCoA, we were able to predict the
k
4
values for PNA to be 0.60, 0.31, 0.89 and 0.26 s
)1
,
for wild-type, Y190F, Y190I and Y190A, respectively.
These k
4
values are similar to the k
cat
values, indicat-
ing that in contrast to the acetylation of PABA by
AcCoA, deacetylation (k
4
) is the rate-limiting step
when PNA is the acetyl acceptor.
PNPA as the acetyl donor; k
cat

¼ k
4
ð1Þ
AcCoA as the acetyl donor; k
cat
¼ k
2
k
4
= k
2
þ k
4
ðÞð2Þ
Because the arylamine substrates possess different
pK
a
values, we further quantified the effect of the sub-
strate’s pK
a
on k
4
by constructing a Brønsted plot. This
is shown in Fig. 4. Previously, the most dramatic feature
of the Brønsted plot for wild-type NAT2 was that
although log (k
4
) shows a good correlation with the
conjugate acid pK
a

values of the arylamines (pK
NH3+
)
and pK
H3O+
, ranging from )1.7 to 4.67, the most basic
substrate, anisidine (pK
NH3+
5.34), exhibits a lower
reactivity than PABA (pK
NH3+
4.67) [27]. This unusual
rate decrease found for anisidine was previously
rationalized as a mechanism shift from rate-limiting
nucleophilic attack by the arylamine to deprotonation
of a tetrahedral intermediate, occurring almost precisely
at the pK
a
of the active site His [27]. In contrast to
wild-type NAT2, the Brønsted plot for Y190I, Y190A
AB
CD
Fig. 3. pH dependence of transacetylation
of hamster NAT2 with PNPA ⁄ PABA.
+
k
1
k
–1
E

pNP
AcE + ArNH
2
k
2
E
E
PNPA
PNP
E A
or or
AcCoA
AcCoA
or
CoASH
AcE
k
3
k
–3
ArNH
2
k
4
AcArNH
2
Scheme 2. Transacetylation of PNPA or AcCoA by NAT.
X. Zhou et al. The catalytic role of Y190 in NAT
FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6933
and Y190F clearly demonstrated an altered dependence

of the reaction on the pK
a
and nucleophilicity of the
acceptor amine. Smaller b
nuc
values were observed
(b
nuc
= 0.6 ± 0.1 for Y190F, b
nuc
= 0.4 ± 0.1 for
Y190I, b
nuc
= 0.5 ± 0.1 for Y190A) for the Brønsted
plot for pK
NH3+
(or pK
H3O+
) ranging from )1.7 to 5.34
(Fig. 4). These results indicate that for the mutants, less
proton transfer occurs during the transition state as
compared with the wild-type, and there is less bond for-
mation between the nitrogen and the thioester carbonyl
than occurs for the wild-type. In addition, because the
increase in pK
a
transacetyl
for Y190I and Y190A approxi-
mates the anisidine pK
a

, the reaction shifts for these
mutants from being dominated by the deprotonation of
a tetrahedral intermediate (Scheme 3, TS-II) to nucleo-
philic attack of the thioester (Scheme 3, TS-I).
For anisidine, deprotonation must occur by Y190F
after formation of the tetrahedral intermediate,
because the pK
a
transacetyl
for His107 (5.48 ± 0.06) is
lower than that of anisidine. Consequently, as
observed for the wild-type NAT catalytic mechanism,
the catalytic mechanism of PABA transacetylation for
the Y190F mutant depends on deprotonation of the
incoming arylamine before formation of the tetra-
hedral intermediate.
Discussion
The essential Cys-His-Asp catalytic triad in NAT has
been identified among several prokaryotic and eukary-
otic members. Each member of the triad has been
shown to be crucial for enzymatic activity. The active
site Cys69 (or Cys70) mutants (Ala, Gln, Ser), H110
mutants (Arg, Trp, Ala) and D127 mutants (Trp, Asn,
Ala) of M. smegmatis NAT and S. typhimurium NAT,
although they can be prepared in soluble form, were
totally devoid of enzyme activity [25]. In contrast, the
unavailability of active mutants of hamster NAT2 at
His107 and Asp122 after refolding suggested that these
two catalytic residues have both catalytic and struc-
tural roles [26,27].

With the exception of the catalytic triad, little is
known about the role of other active site residues on
eukaryote NAT catalysis and binding. X-ray crystallo-
Table 2. Steady-state kinetics data for transacetylation by wild-type and Y190 mutants at 25 °C and pH 7.0.
Hamster
NAT2
K
a
(mM)
K
b
(mM)
k
cat
(s
)1
)
k
cat
⁄ K
a
(s
)1
ÆmM
)1
)
k
cat
⁄ K
b

(s
)1
ÆmM
)1
)
PNPA ⁄ anisidine
OCH
3
H
2
N
pKa = 5.34
Wild-type
a
72.8 ± 0.4 0.34 ± 0.04 260 ± 20 100 ± 20 790 ± 120
Y190F 7.8 ± 0.6 0.58 ± 0.04 288 ± 14 37 ± 4 496 ± 58
Y190I 10.1 ± 1.5 0.18 ± 0.03 85 ± 12 8.4 ± 2.4 483 ± 160
Y190A 7.4 ± 0.5 0.25 ± 0.02 28 ± 2 3.8 ± 0.6 114 ± 16
PNPA ⁄ PABA
COOHH
2
N
pKa = 4.67
Wild-type
a
10 ± 1 0.23 ± 0.02 620 ± 40 62 ± 9 2700 ± 400
Y190F 7.7 ± 0.9 0.22 ± 0.01 393 ± 29 51 ± 10 1786 ± 214
Y190I 4.5 ± 0.7 0.08 ± 0.01 60 ± 4 13 ± 3 746 ± 146
Y190A 7.5 ± 0.5 0.11 ± 0.01 38 ± 2 5 ± 1 345 ± 46
PNPA ⁄ pABglu

H
2
N
CH
2
CH
2
COOH
O
HN
COOH
pKa = 2.93
Wild-type
a
1.5 ± 0.1 1.7 ± 0.1 120 ± 3 86 ± 3 70 ± 5
Y190F 2.8 ± 0.2 2.5 ± 0.2 86 ± 3 30 ± 3 34 ± 4
Y190I 11 ± 1 2.7 ± 0.2 77 ± 4 7 ± 1 28 ± 4
Y190A 5.6 ± 0.6 6.2 ± 0.6 16 ± 1 2.8 ± 0.5 2.6 ± 0.4
AcCoA ⁄ PNA
NO
2
H
2
N
pKa = 1
Wild-type
a
0.037 ± 0.003 0.77 ± 0.06 0.60 ± 0.02 16 ± 2 0.78 ± 0.08
Y190F 0.14 ± 0.03 0.48 ± 0.10 0.31 ± 0.03 2.23 ± 0.79 0.66 ± 0.21
Y190I 0.71 ± 0.18 1.51 ± 0.44 0.89 ± 0.15 1.26 ± 0.54 0.60 ± 0.27

Y190A 1.49 ± 0.8 2.92 ± 1.64 0.25 ± 0.11 0.17 ± 0.16 0.087 ± 0.086
AcCoA ⁄ PABA Wild-type
a
3.4 ± 0.3 0.12 ± 0.01 200 ± 1 60 ± 6 1700 ± 200
Y190F 1.8 ± 0.2 0.14 ± 0.02 189 ± 17 106 ± 15 1360 ± 226
Y190I 1.9 ± 0.6 0.06 ± 0.01 58 ± 9 30 ± 13 974 ± 377
Y190A 2.7 ± 0.9 0.28 ± 0.10 8.14 ± 2.23 3 ± 1 29 ± 11
a
Values for the ‘wild-type’ protein are taken from [27].
The catalytic role of Y190 in NAT X. Zhou et al.
6934 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
graphic analysis revealed that the para-hydroxyl moi-
ety of Tyr190, which resides at a b sheet that is close
to the 17-residue insertion loop (163–187) (Fig. 1A), is
positioned within the active site hydrophobic core,
where the hydroxyl group forms a hydrogen bond with
Asp122 of the catalytic triad (Fig. 1). This Tyr190 is
highly conserved across prokaryotic and eukaryotic
NATs [34,35], with the only exception being the trun-
cated banatB isoform from Bacillus anthracis, where a
His is at the equivalent position [36]. Closer inspection
revealed that in addition to the side chain of Tyr190,
the side chain of Asn72 and the backbone of Gly124
and Ala123 participate in a network of interactions
with Asp122. Moreover, because the centroid of the
Tyr190 phenyl ring is $ 3.5 A
˚
from the centroid of the
His107 imidazole ring and the planes of the two ring
systems intersect at an angle of $ 30°, Tyr190 and

His107 interact by a common aromatic stacking inter-
action. To gain insight into the role of Tyr190 on
NAT catalysis, we characterized a set of point site
mutants at this position by steady-state and presteady-
state kinetics and NMR spectroscopy.
Unlike His107 and Asp122, mutations at the 190 posi-
tion in hamster NAT2 neither affect the protein’s overall
folding and stability nor abolish the enzymatic activity,
indicating that hamster NAT2 is flexible enough to
accommodate such alterations at the point site. On the
other hand, the Tyr to Phe substitution is considered to
be a relatively conservative substitution [40], whereas
the Tyr to Ile substitution is expected to maintain the
secondary structure, as b strand formation is favored by
Ile [41]. Therefore, these two mutants were designed in
order to minimize structural perturbation. In contrast,
the Tyr to Ala conversion would be expected to impact
catalysis, as replacement of a phenol side chain with a
methyl group eliminates hydrophobic packing interac-
tions proximal to the active site.
Our finding that the conservative mutation of
hamster NAT2 Y190F modestly diminishes the k
cat
value for transacetylation of PABA by PNPA provides
supporting kinetic evidence for the similarity of the
Tyr190 to Phe mutant and wild-type. However,
the rate of acetylation of NAT2 (k
2
) is 5-fold
lower than the wild-type, and the stability of the

Scheme 3. Proposed transition states of the NAT-catalyzed trans-
acetylation reaction [27].
A B
C D
Fig. 4. Brønsted plots of the deacetylation rate constants for the acetyl-enzyme with various arylamine substrates (k
4
) and H
2
O(k
H2O
). (A)
Wild-type. Values for the ‘wild-type’ protein are taken from [27]. Linear regression of the data resulted in the line with the slope
b
nuc
= 0.8 ± 0.1 and r
2
= 0.97 for the five substrates, except anisidine. (B) Y190F. Linear regression of the data resulted in the line with the
slope b
nuc
= 0.6 ± 0.1 and r
2
= 0.93 for the five substrates. (C) Y190I. Linear regression of the data resulted in the line with the slope
b
nuc
= 0.4 ± 0.1 and r
2
= 0.85 for the five substrates. (D) Y190A. Linear regression of the data resulted in the line with the slope
b
nuc
= 0.5 ± 0.1 and r

2
= 0.92 for the five substrates.
X. Zhou et al. The catalytic role of Y190 in NAT
FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6935
acetylated enzyme intermediate is affected, which can
be attributed to the removal of the hydrogen bond
between the Tyr hydroxyl group and the aspartyl car-
bonyl group (Table 1). Therefore, the modest decrease
in the k
cat
values for transacetylation of PNPA ⁄ PABA
suggests that the role of the hydroxyl group (i.e.
H-bonding) of Tyr190 in hamster Y190F is masked by
the turnover number, which is mainly affected by k
4
rather than k
2
. In contrast, the significant loss of cata-
lytic efficiency for the Y190I and Y190A mutants is
supportive of a potential role in catalysis played by the
imidazole–aromatic interaction between Y190 and
H107 (Fig. 1) [41,42]. Loewenthal et al. [43] found that
the aromatic–His interaction in barnase stabilizes the
protonated His, increasing its pK
a
value and, therefore,
increasing the nucleophilicity of active site Cys. A simi-
lar interaction was found between the indole ring of
an active site Trp177 and the imidazole of the catalytic
triad His159 in the papain-like Cys proteinase [44].

Mutations of the Trp to either Tyr, Phe, Ile or Ala
(the strength of the His–aromatic interaction decreases
in the series His-Trp greater than His-Tyr greater than
His-Phe) lead to elevation of the Cys pK
a
and destabi-
lization of the thiolate–imidazolium ion pair [44]. Simi-
larly, pK
a1
acetyl
, which has been associated with Cys68
for hamster NAT2 [26], was raised by approximately
one pK
a
unit when Tyr190 was replaced with the
aliphatic amino acid, Ile or Ala.
Although replacement of Tyr190 with Phe seems to
have little effect on the maximum turnover number, the
altered pH profiles of acetylation and transacetylation
underscore the importance of the hydroxyl group and
raise several points of interpretation. First, as can be
seen from the pH versus rate of acetylation profiles,
Y190F exhibited different levels of dependence from
that of the wild-type; nevertheless, the first inflection
point is similar, corresponding to the pK
a
acetyl
of the
active site Cys. This unchanged pK
a

acetyl
of the active
site Cys in Y190F could be ascribed to dipole–dipole
interaction between the para-hydrogen of Phe and the
aspartyl oxygen that stabilizes the formation of the thio-
late–imidazolium ion pair through Asp122, albeit less
efficiently than the Tyr hydroxyl [45,46]. Second, it is
problematic to assign the second pK
a
acetyl
for acetylation
of the wild-type (pK
a2
acetyl
6.79 ± 0.25) to the ioniza-
tion of the hydroxyl group in Y190. It is tempting
to assign this pK
a2
acetyl
to Y190, as this pK
a2
acetyl
is
absent from the profile for Y190F. However, the second
pK
a
acetyl
emerges for both the Y190I and Y190A
mutants. Thus, it is more likely that pK
a2

acetyl
reflects
ionization of a pH-sensitive residue that indirectly
affects conformation of the active site, as no putative
ionizable side chain responsible for this pK
a2
acetyl
appears in the active site. The lower reactivity of the
active site Cys in Y190I and Y190A is consistent with
the elevated pK
a1
acetyl
from k
2
⁄ K
m
acetyl
versus pH, as
these side chains probably raise the pK
a
of Asp122 and
thus Cys68.
Although considerably different from the wild-type
profile, the pH rate profiles for transacetylation for the
three mutants with PNPA ⁄ PABA were similar to each
other. The pK
a1
transacetyl
of Y190F was similar to the
wild-type, whereas the pK

a1
transacetyl
values of Y190I
and Y190A were about one unit higher. Under the
assumption that, like wild-type NAT2, the Y190
mutants utilize general base catalysis and His107 corre-
sponds to the first pK
a1
transacetyl
, the experimental data
are consistent with our previously proposed model [27].
The pK
a1
transacetyl
increase in His107 enhances the ability
of the base to deprotonate the attacking arylamine
before a positive charge is developed on the arylamine.
Previously, we have shown that the pK
a1
transacetyl
of the
active site His (5.55 ± 0.14) is matched to that of
PABA (pK
a
= 4.67), thus facilitating concerted depro-
tonation of the incoming arylamine nucleophile in the
transition state (Scheme 3, TS-I). If, however, the
pK
a
transacetyl

of the His is significantly lower than that of
the conjugate acid of the attacking arylamine, then
deprotonation is favored to follow the tetrahedral inter-
mediate formation (Scheme 3, TS-II). Consequently, as
demonstrated by the Brønsted plots, deacetylation of
the acetylated Y190I and Y190A mutants results in
more efficient acetylation of anisidine (pK
a
= 5.34), as
deprotonation is more favored to occur concomitantly
with arylamine attack at the thioester carbonyl.
The observation of the second pK
a
transacetyl
for the
pH rate profile for transacetylation by all three
mutants is problematic, as it would be expected that
the altered pK
a1
transacetyl
of active site His would be
matched by that of the associated altered Asp122. To
address this issue we carried out protein NMR struc-
tural studies of the most altered mutant, Y190A. The
results of those studies revealed an altered active site,
including Asp122 and the most closely associated inner
sphere side chains. Consequently, we propose that
Y190 probably functions not only as a hydrogen bond
donor to Asp122, but also as a ‘damper’ of the inher-
ent sensitivity of the active site to undergo reorganiza-

tion. Recently, the importance of protein dynamics on
catalysis has become increasingly apparent [47,48]. The
backbone dynamics of hamster NAT2 has been char-
acterized by NMR experiments, with slower, low-fre-
quency motions, detected for the active site cavity [49].
In contrast, faster motions were found for the regions
spanning N177–L180 and D285–F288, leading to a
proposal that these residues act as a ‘gate-like’
The catalytic role of Y190 in NAT X. Zhou et al.
6936 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
structure to accommodate substrate interaction [49].
Our results with NAT suggest that the role of some
residues may not be just to enhance catalytic efficiency
by facilitating productive protein dynamic states, but
also to reduce the occurrence of unproductive modes
over a variety of environmental conditions, such as
pH, thus increasing catalytic robustness. Whereas most
catalytically impaired NAT polymorphisms result from
highly destabilizing mutations on gene product trunca-
tions, the availability of the Tyr190 mutants makes it
feasible to conduct cell-based studies of the effects of
the stability of the acetylated enzyme intermediate on
the N-acetylation of aromatic amines, on the bioactiva-
tion of N-arylhydroxylamines by O-acetylation to pro-
duce DNA adducts, and on the intracellular fate of the
NAT protein [50,51].
Experimental procedures
Materials
AcCoA, PABA, PNPA, ampicillin, anisidine, Mops, 3,3-
dimethylglutaric acid, pABglu and PNA were purchased

from Sigma-Aldrich (St Louis, MO, USA). BL21 Codon
Plus (RIL) competent Escherichia coli cells were purchased
from Stratagene (La Jolla, CA, USA). DEAE Sepharose
Fast Flow anion-exchange resin was purchased from Amer-
sham Pharmacia (Ann Arbor, MI, USA). Steady-state
kinetic data were collected on a Varian Cary 50 UV–visible
spectrophotometer (Palo Alto, CA, USA). Transient kinetic
data were obtained on a single-wavelength stopped-flow
apparatus (Applied Photophysics, Leatherhead, UK, model
SX.18MV). Kinetic data were analyzed with the jmp in 4
software (SAS Institute, Inc., Cary, NC, USA)
Site-directed mutagenesis, protein expression
and purification
Site-directed mutagenesis of the hamster NAT2 Tyr190 to
Phe (Y190F), to Ile (Y190I) and to Ala (Y190A), was
carried out using the pPH70D vector and QuickChange
site-directed mutagenesis kit (Stratagene)[52]. The oligonu-
cleotide primers used for Y190F, Y190I and Y190A were
5¢-GA AAG ATC TTT
190
TCT TTT ACT CTT GAA
CCC CG-3¢,5¢-GA AAG ATC ATT
190
TCT TTT ACT
CTT GAA CCC CG-3¢ and 5¢-GA AAG ATC GCT
190
TCT TTT ACT CTT GAA CCC CG-3¢, respectively. The
automated DNA sequencing results showed that the desired
sites of mutations had been achieved. The mutated plasmids
were transformed to BL21 Codon Plus (RIL) E. coli cells

according to the protocol of the manufacturer.
The expression and purification of the mutants were
similar to those for wild-type hamster NAT2 as previ-
ously described [52]. Overnight cultures (10 mL) were
grown from single colonies and were diluted to 1 L ter-
rific broth containing ampicillin (100 lgÆmL
)1
) and chl-
oramphenicol (50 lgÆmL
)1
). Cultures were grown at
37 °C to an absorbance (A
600
) of 0.6, at which time iso-
propyl thio-b-d-galactoside was added to a final concen-
tration of 0.2 mm. After isopropyl thio-b-d-galactoside
induction, cells were incubated for an additional 17 h of
growth at 17 °C and harvested. The cell pellets were
lysed as previously reported [52]. The mutated NAT2–di-
hydrofolate reductase fusion proteins were purified by an
ion exchange column (50 mm diameter) packed with Q-
Sepharose fast flow beads (Pharmacia, 60 mL) and eluted
from the column at 0.26 m KCl. The dihydrofolate reduc-
tase–NAT2 fusion proteins subsequently underwent
human thrombin cleavage and were applied to the second
Q-Sepharose column. NAT2 was eluted at 0.08 m KCl.
Both columns were coupled with a Pharmacia FPLC sys-
tem with an LCC 500 plus system controller, two P500
solvent delivery pumps and a P500 collector. Protein con-
centrations were determined with the Bradford protein

assay [53].
NAT2 activity assay
The specific activity of wild-type and mutant NAT2 was
measured using PNPA as the acetyl donor and PABA as
the acetyl acceptor in Mops buffer (pH 7, 25 °C), as
described previously [27]. The reaction buffer contained
0.5 lgÆmL
)1
NAT2, 0.5 mm PABA and the reaction was
initiated by adding PNPA in dimethylsulfoxide (final
concentration 2 mm, dimethylsulfoxide 1%). The rate of
the reaction was determined by monitoring the linear
increase in absorbance at 400 nm because of the formation
of PNP. The specific activity was calculated and expressed
in lmÆmg
)1
Æmin
)1
.
Presteady-state kinetic parameters for the
acetylation of NAT
The single turnover reactions of the acetylation of NAT2
were monitored at 25 °C using a single wavelength
stopped-flow apparatus (Applied Photophysics, model
SX.18MV). PNPA (160–3000 lm) in Mops buffer [1 mL,
100 mm; 150 mm NaCl, and 3% dimethylsulfoxide (pH
7.0)] was transferred to one stopped-flow syringe. NAT2
(Y190F, 276 lgÆmL
)1
,8lm; Y190I 353 lgÆmL

)1
, 10.2 lm;
Y190A 642 lgÆmL
)1
, 18.6 lm) in Mops buffer [1 mL,
100 mm; with 150 mm NaCl (pH 7.0)] was transferred to
the second stopped-flow syringe. Each time equal volumes
(50 lL) of the enzyme solution and the substrate were
injected and mixed rapidly. The production of PNP [P] was
monitored at 400 nm [42]. The single turnover timecourse
curves were fitted with Eqn (3) using jmp in 7 software,
where A is the amplitude and k
obs
is the pseudo-first-order
rate constant for the acetylation step. The results represent
X. Zhou et al. The catalytic role of Y190 in NAT
FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6937
the average of three experiments. The kinetic parameter
k
2
⁄ K
m
acetyl
was obtained by plotting k
obs
versus PNPA con-
centration (Eqn 4) (Fig. S3).
PtðÞ¼A 1 À e
Àk
obs

Át
ÀÁ
ð3Þ
k
obs
¼
k
2
 PNPA½
K
acetyl
m
þ PNPA½
ð4Þ
Steady-state kinetics of acetyl-enzyme hydrolysis
Hamster NAT2 (0, 1, 2, 4 and 8 lm) in Mops buffer
[100 mm; with 150 mm NaCl (pH 7.0) and 0.1 mm dith-
iothreitol] was added to PNPA (final concentration 320 lm)
to a total 500 lL volume [26]. The reaction rate was deter-
mined at 25 °C by measuring the linear increase in absor-
bance at 400 nm during the initial 5 min. Control
experiments were carried out in the absence of NAT2. The
slope of the linear increase in absorbance at 400 nm repre-
sents the velocity of the hydrolysis of the acetyl-enzyme
intermediate (V). The acetylated enzyme hydrolysis rate
constant, k
hydrolysis
, can be calculated from Eqn (5), where
the values of k
2

, K
m
acetyl
were obtained from presteady-state
kinetics and the values of V, E
total
, and [PNPA] were from
the experiments. Subsequently, the half-life of the acetyl-
enzyme intermediate, T
1 ⁄ 2
, was calculated from Eqn (6).
E
tot
V
¼
K
acetyl
m
k
2
PNPA½
þ
1
k
2
þ
1
k
hydrolysis
ð5Þ

T
1=2
¼ Ln2=k
hydrolysis
ð6Þ
Steady-state kinetic parameters for
transacetylation of arylamine substrate
As described previously, the assay was performed using
PNPA or AcCoA as the acetyl donor and one of the fol-
lowing primary arylamines as the acetyl acceptor: anisi-
dine, PABA, pABglu or PNA [27]. The reaction rates
were determined in triplicate at 25 °C at a fixed concen-
tration of one substrate while varying the concentration of
the other substrate. All the kinetic parameters were deter-
mined with at least five different concentrations of each
substrate.
The initial velocities of the reaction with PNPA ⁄ anisi-
dine, PNPA ⁄ PABA and PNPA ⁄ pABglu were measured as
the linear increase in the absorbance at 400 nm due to the
formation of PNP (e
400nm
= 9400 m
)1
Æcm
)1
). In a final vol-
ume of 500 lL, NAT2 (Y190F 0.3 lgÆmL
)1
, 8.76 nm;
Y190I 1 lgÆmL

)1
,28nm; Y190A 3 lgÆmL
)1
,88nm) was
incubated with either anisidine (0.025–1.6 mm), PABA
(0.05–1.2 mm), pABglu (0.25–12 mm) or PNA (0.5–8 mm)
in Mops buffer (100 mm at pH 7.0, 150 mm NaCl, and
0.1 mm dithiothreitol). The reactions were initiated by the
addition of PNPA dissolved in dimethylsulfoxide (20 lL).
The final concentration of dimethylsulfoxide was 4%.
The initial velocity of the reaction with AcCoA ⁄ PABA
was measured as a linear decrease in PABA concentration.
In a final volume of 1000 l L dimethylglutaric acid buffer
(50 mm,80mm NaCl and 0.1 mm dithiothreitol at pH 7.0),
NAT2 (Y190F 0.3 lgÆmL
)1
, 8.76 nm; Y190I 0.5 lgÆmL
)1
,
14 nm; Y190A 1.5 lgÆmL
)1
,44nm) was incubated with Ac-
CoA (0.125–8 mm) for $ 1 min, then the reaction was initi-
ated by adding PABA (0.03–0.4 mm). Aliquots (20–120 l L)
of the reaction mixture were withdrawn at 10–20 s intervals
(0–200 s) and transferred to an assay mixture containing
trichloroacetic acid (4%, v ⁄ v), 4-(dimethylamino)benzalde-
hyde (2.5%, w ⁄ v), and acetonitrile (45%, v ⁄ v) (final volume
300 lL). The residual PABA was quantified by measuring
the absorbance at 450 nm of the formation of Schiff base

(e
450nm
= 52835 m
)1
Æcm
)1
) [54].
The initial velocity of the reaction with AcCoA ⁄ PNA was
measured as a linear decrease in the absorbance at 430 nm
because of the acetylation of PNA (e
430nm
= 3298 m
)1
Æcm
)1
).
In a final volume of 300 lL, NAT2 (Y190F 0.3 lgÆmL
)1
,
8.76 nm; Y190I 1 lgÆmL
)1
,28nm; Y190A 3 lgÆmL
)1
,88nm)
was incubated with AcCoA (10 lL, final concentration 0.04–
0.8 mm) and PNA (0.05–2.5 mm) in dimethylglutaric acid
buffer (50 mm,80mm NaCl and 0.1 mm dithiothreitol at pH
7.0). The reactions were monitored over a maximum of 5 min.
pH dependence of acetylation of NAT
At pH values ranging from 5.2 to 9.0, the kinetic parameter,

k
2
⁄ K
m
acetyl
, was determined using stopped-flow apparatus
with NAT2 (final concentration Y190F 4 lm, Y190I 10 lm,
Y190A 19 lm) and PNPA (final concentration 800 lm).
Either dimethylglutaric acid buffer (50 mm at pH 5.0–7.0,
0.1 mm dithiothreitol) or Tris buffer (50 mm at pH 7.5–9.0,
0.1 mm dithiothreitol) was used. The ionic strength was
held constant at 150 mm with NaCl. Assays were performed
in triplicate as described for the presteady-state kinetic
experiments ( vide supra), except that the formation of PNP
was monitored at 340 nm for pH 5.2–6 and at 400 nm for
pH 6.4–9. The single turnover timecourse curves were fitted
with Eqn (3) using jmp in 4 software, where the pseudo-first-
order rate constant, k
obs
, for the acetylation step was
abstracted, and the parameter k
2
⁄ K
m
acetyl
was obtained.
To fit the k
2
⁄ K
m

acetyl
versus pH data for the wild-type,
Y190I and Y190A, Eqn (8) was used; for Y190F Eqn (7)
was used.
k
2
=K
acetyl
m
ÀÁ
k
2
=K
acetyl
m

lim
¼
1 þ r Á 10
pKaÀpHðÞ
1 þ 10
pKaÀpHðÞ
ð7Þ
k
2
=K
acetyl
m
ÀÁ
k

2
=K
acetyl
m

lim
¼
1 þ r Á 10
pHÀpKa2ðÞ
1 þ 10
pHÀpKa2ðÞ
þ 10
pKa1ÀpHðÞ
ð8Þ
The catalytic role of Y190 in NAT X. Zhou et al.
6938 FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS
pH dependence of transacetylation of NAT with
PNPA and PABA
At pH values ranging from 5.2 to 9.0, the kinetic parameters,
k
cat
⁄ K
PABA
, were determined at a fixed, saturated concentra-
tion of PNPA (2 mm) and six concentrations of PABA (10–
120 mm) with NAT2. Either dimethylglutaric acid buffer
(50 mm at pH 5.0–7.0, 0.1 mm dithiothreitol) or Tris buffer
(50 mm at pH 7.5–9.0, 0.1 mm dithiothreitol) was used. The
ionic strength was held constant at 150 mm with NaCl.
Assays were performed as described in previous steady-state

kinetics, except that the formation of PNP was monitored at
the isosbestic point of 349 nm (e
349nm
= 5700 m
)1
Æcm
)1
). To
fit the k
2
⁄ K
PABA
versus pH data for the wild-type, Eqn (9)
was used; for Y190F, Y190I and Y190A, Eqn (10) was used.
k
cat
=K
PABA
ðÞ
k
cat
=K
PABA
ðÞ
lim
¼
1 þ r Á 10
pKaÀpHðÞ
1 þ 10
pKaÀpHðÞ

ð9Þ
k
cat
=K
PABA
ðÞ
k
cat
=K
PABA
ðÞ
lim
¼
1 þ r Á 10
pHÀpKa2ðÞ
1 þ 10
pHÀpKa 2ðÞ
þ 10
pKa1ÀpHðÞ
ð10Þ
CD spectroscopy and HSQC of
15
N-labeled Y190A
and wild-type NAT2
For CD spectroscopy, 3 lm NAT2 protein samples were dis-
solved in 10 mm potassium phosphate (pH 7) containing
0.5 mm EDTA and 0.05 mm dithiothreitol. Spectra were
acquired at 25 °C on a Jasco J-710 spectropolarimeter. For
HSQC experiments,
15

N-labeled wild-type and Y190A ham-
ster NAT2 were prepared by growth in M9 minimal medium
with
15
N-labeled ammonium chloride (1 gÆL
)1
M9 minimal
medium) as the only nitrogen source [34,52]. The purified
15
N-
labeled protein was concentrated to 0.2 mm in NMR buffer
[30 mm sodium phosphate, 50 mm NaCl, 4 mm dithiothreitol,
pH 6.8, 0.1% (w ⁄ v) NaN
3
, 10% D
2
O]. HSQC spectra were
acquired on Varian 600 MHz spectrometers equipped with a
Bioselect probe at 10 °C, as described previously [34].
Acknowledgements
This work was funded by grants from the Leukemia
Research Foundation (CRW), Developmental Grants
for Drug Design, the Department of Medicinal Chemis-
try, University of Minnesota (CRW, PEH), the National
Institutes of Health (CA117888 to KJW) and American
Cancer Society (RSG-07-186-01-GMC to KJW). We
thank Drs Tsui-Fen Chou and Haiqing Wang for their
valuable help in experiments and discussions.
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Supporting information
The following supplementary information is available:
Fig. S1. CD of wild-type and Y190 mutants at pH 7 at
room temperature.
Fig. S2. (A) Superposition of [
1
H,
15
N] HSQC spectra
of wild-type hamster NAT2 (black) and Y190A
hamster NAT2 (red). (B) Affected residues in Y190A
hamster NAT2.
Fig. S3. Presteady-state kinetics of formation of the
acetyl-enzyme intermediate by wild-type and Y190
mutants.
This supplementary material can be found in the

online version of this article.
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X. Zhou et al. The catalytic role of Y190 in NAT
FEBS Journal 276 (2009) 6928–6941 ª 2009 The Authors Journal compilation ª 2009 FEBS 6941