Tyr190 was also shown to play an important role in determining the pKa of the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation.. Results
Trang 1N-acetyltransferase 2 catalytic triad by site-directed
mutagenesis of the proximal conserved residue, Tyr190 Xin Zhou1, Naixia Zhang2, Li Liu1, Kylie J Walters2, Patrick E Hanna1 and Carston R Wagner1
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-acetyN-acety-lation 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; pKa
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: wagne003@umn.edu
(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 pKa 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 [1H, 15N] 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.
Trang 2in 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 [2-3H] 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 pKaof 5.2 [26]
How-ever, in contrast to Cys proteases, which typically
exhibit an additional basic limb pKaof 8–9, the second
pKa for hamster NAT2 acetylation was found to be
> 9.5 For both NATs and Cys proteases, the basic
pKahas 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 catacata-lytic 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.
Trang 3sized 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
15N-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 [1H,15N] heteronuclear
single quantum coherence (HSQC) experiments to
record the chemical shift values of NAT amide
nitro-gen and hydronitro-gen atoms 15N-labeled proteins were
prepared and [1H,15N] 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)1for 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, k2⁄ Kmacetyl, for the Y190 mutants was 2–20-fold lower than the value observed for the wild-type (Table1), indicating a slower rate of enzyme acetylation The decrease in the k2⁄ Kmacetyl value was largely due to a decrease in k2, rather than a significant change in Kmacetyl In the case of the Y190F mutant, the value of k2 decreased slightly from
1301 ± 716 s)1(wild-type) to 279 ± 54 s)1; however, a pronounced k2 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
Trang 4through rapid formation of an enzyme intermediate
(k2) followed by rate-limiting hydrolysis (khydrolysis) [26]
(Scheme1) 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 (khydrolysis) 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
(k2) by 4.7-fold and an increase in the rate of
interme-diate hydrolysis (khydrolysis) of 3.6-fold Similarly, a
decrease in k2 and an increase in khydrolysis ( 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 k2
was observed However, the value of khydrolysis 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 deacetylaacetyla-tion, perturbaacetyla-tion of the active site
by complete removal of the side chain mainly affected
enzyme acetylation (k2)
pH dependence of NAT acetylation
Usually, the pH dependence of acetylation of NAT
(k2⁄ Kmaceytl) 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,
k2⁄ Kmacetyl, fit best to a model for two pKa values, with the first, pKa1acetyl (5.16 ± 0.14), assigned to the active site Cys, and the second, pKa2acetyl (6.79 ± 0.25), assigned to a probable conformational change [26] In the case of Y190F, the value of log (k2⁄ Kmacetyl) rose as a function of pH until a plateau was reached above pH 7.5 The data were fit into a one-pKa model with a pKaacetyl value of 5.16 ± 0.05, which is virtually identical to the first pKa1acetyl (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 pKa, which is consistent with the slightly reduced acetylation rate k2 However, in contrast with the pH profile for wild-type NAT2, where the maximum k2⁄ Kmacetyl was reached at pH 6.4, the
k2⁄ Kmacetyl 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 pKaacetyl, 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-pKa model with the pKa1acetyl 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 (kcat⁄ KPABA) 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
k1
k–1 E PNPA
pNP AcE + H 2 O
k2
AcOH
khydrolysis
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 macetyl
k 2 ⁄ K macetyl
( M )1Æs)1)
khydrolysis (s)1)
T 1 ⁄ 2
(s)
a Values for the ‘wild-type’ protein are taken from [26].
Trang 5step For wild-type NAT2, the pH influence on
kcat⁄ KPABA revealed only one pKatransacetyl at
5.55 ± 0.14 with two active forms and a (kcat⁄
KPABA)lim of 3000 ± 50 mm)1Æs)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-pKa model with two active forms,
and decreasing (kcat⁄ KPABA)lim values (Fig 3) In our
previous solvent isotope effect study of wild-type
NAT2, a normal solvent kinetic isotope effect
[H⁄ D(kcat⁄ Kb)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
pKatransacetyl of 5.55 for the acetylated enzyme [27]
We assumed general base catalysis was also
employed by the Y190 mutants, thus, the first
pKa1transacetyl 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 pKa1transaccetyl of
5.48 ± 0.06, which is similar to the pKatransacetyl of
the wild-type, and consistent with a transacetylation
rate similar to the wild-type In contrast, the
pKa1transacetyl 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 (k4) (Scheme2) from acetylated NAT2 proceeds much faster (1000–10 000-fold) than hydrolysis of the acetylated NAT intermedi-ate (khydrolysis) [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 (Table2) Previously we have shown that for reac-tions with PNPA as the acetyl donor, the transacetyla-tion of arylamine substrate (k4), rather than the acetylation of NAT2 (k2), is the rate-limiting step [27] Therefore, the kcat values for PNPA⁄ anisidine, PNPA⁄ PABA, PNPA ⁄ pABglu approximate k4 (the rate of transacetylation of amine acceptors) (Eqn 1)
Fig 2 pH dependence of hamster NAT2 single turnover by PNPA.
Trang 6However, for reactions employing AcCoA as the acetyl
donor, the kcatvalues are determined by the individual
rate constants for both the acetylation (k2) and
deacet-ylation (k4) steps (Eqn 2) [27] On the basis of the
Ping Pong mechanism, the acetylation rate (k2) of
NAT2 by the acetyl donor is independent of the
trans-acetylation rate (k4) of the arylamine substrate
Because the values of k4 for PABA can be inferred
from the PNPA⁄ PABA reaction, which are in the
range of 38–620 s)1, based on the kcat values for
PABA acetylation by AcCoA (Eqn 2), the values of k2
for AcCoA can be predicted to range from 10 to
1740 s)1 Hence, from the kcat values for the
acetyla-tion of PNA by AcCoA, we were able to predict the
k4 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 k4 values are similar to the kcat values,
indicat-ing that in contrast to the acetylation of PABA by
AcCoA, deacetylation (k4) is the rate-limiting step
when PNA is the acetyl acceptor
PNPA as the acetyl donor; kcat¼ k4 ð1Þ AcCoA as the acetyl donor; kcat¼ k2k4=ðk2þ k4Þ ð2Þ Because the arylamine substrates possess different
pKa values, we further quantified the effect of the sub-strate’s pKaon k4by constructing a Brønsted plot This
is shown inFig 4 Previously, the most dramatic feature
of the Brønsted plot for wild-type NAT2 was that although log (k4) shows a good correlation with the conjugate acid pKa values of the arylamines (pKNH3+) and pKH3O+, ranging from)1.7 to 4.67, the most basic substrate, anisidine (pKNH3+ 5.34), exhibits a lower reactivity than PABA (pKNH3+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 pKa of the active site His [27] In contrast to wild-type NAT2, the Brønsted plot for Y190I, Y190A
Fig 3 pH dependence of transacetylation
of hamster NAT2 with PNPA ⁄ PABA.
+
k1
k–1
E
pNP AcE + ArNH 2
k2
E
E
or or
or
CoASH
AcE
k3
k4
AcArNH 2
Scheme 2 Transacetylation of PNPA or AcCoA by NAT.
Trang 7and Y190F clearly demonstrated an altered dependence
of the reaction on the pKa and nucleophilicity of the
acceptor amine Smaller bnuc values were observed
(bnuc = 0.6 ± 0.1 for Y190F, bnuc= 0.4 ± 0.1 for
Y190I, bnuc= 0.5 ± 0.1 for Y190A) for the Brønsted
plot for pKNH3+(or pKH3O+) 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 pKatransacetylfor Y190I and Y190A
approxi-mates the anisidine pKa, the reaction shifts for these
mutants from being dominated by the deprotonation of
a tetrahedral intermediate (Scheme3, 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 pKatransacetyl 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
(m M )
K b
(m M )
k cat
(s)1)
k cat ⁄ K a
(s)1Æm M )1)
k cat ⁄ K b
(s)1Æm M )1)
PNPA ⁄ anisidine
OCH3
H2N
pKa = 5.34
PNPA ⁄ PABA
COOH
H2N
pKa = 4.67
PNPA ⁄ pABglu
H2N
CH2CH2COOH
O
HN COOH pKa = 2.93
AcCoA ⁄ PNA
NO2
H2N
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
a Values for the ‘wild-type’ protein are taken from [27].
Trang 8graphic 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 kcat 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 (k2) 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 (k4) and H2O (kH2O) (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 r2= 0.97 for the five substrates, except anisidine (B) Y190F Linear regression of the data resulted in the line with the slope bnuc= 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
bnuc= 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.
Trang 9acetylated 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 kcatvalues 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 k4
rather than k2 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 pKavalue 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 pKa and
destabi-lization of the thiolate–imidazolium ion pair [44]
Simi-larly, pKa1acetyl, which has been associated with Cys68
for hamster NAT2 [26], was raised by approximately
one pKa 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 pKaacetyl of the
active site Cys This unchanged pKaacetyl 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 pKaacetylfor acetylation
of the wild-type (pKa2acetyl 6.79 ± 0.25) to the
ioniza-tion of the hydroxyl group in Y190 It is tempting
to assign this pKa2acetyl to Y190, as this pKa2acetyl is
absent from the profile for Y190F However, the second
pKaacetyl emerges for both the Y190I and Y190A
mutants Thus, it is more likely that pKa2acetyl reflects
ionization of a pH-sensitive residue that indirectly
affects conformation of the active site, as no putative
ionizable side chain responsible for this pKa2acetyl
appears in the active site The lower reactivity of the active site Cys in Y190I and Y190A is consistent with the elevated pKa1acetyl from k2⁄ Kmacetyl versus pH, as these side chains probably raise the pKaof 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 pKa1transacetyl of Y190F was similar to the wild-type, whereas the pKa1transacetyl 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 pKa1transacetyl, the experimental data are consistent with our previously proposed model [27] The pKa1transacetylincrease 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 pKa1transacetylof the active site His (5.55 ± 0.14) is matched to that of PABA (pKa= 4.67), thus facilitating concerted depro-tonation of the incoming arylamine nucleophile in the transition state (Scheme 3, TS-I) If, however, the
pKatransacetylof 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 (pKa= 5.34), as deprotonation is more favored to occur concomitantly with arylamine attack at the thioester carbonyl
The observation of the second pKatransacetyl for the
pH rate profile for transacetylation by all three mutants is problematic, as it would be expected that the altered pKa1transacetyl 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’
Trang 10structure 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-acetylabioactiva-tion 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
CCC CG-3¢, 5¢-GA AAG ATC ATT190
TCT TTT ACT
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
37C to an absorbance (A600) 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 17C 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 syssys-tem 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, 25C), 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)1Æmin)1
Presteady-state kinetic parameters for the acetylation of NAT
The single turnover reactions of the acetylation of NAT2 were monitored at 25C 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, 8 lm; 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 kobsis the pseudo-first-order rate constant for the acetylation step The results represent