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: 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 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 [...]... and 2- aminofluorene J Protein Chem 22 , 631–6 42 24 Dupret JM & Grant DM (19 92) Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2 J Biol Chem 26 7, 7381– 7385 25 Sandy J, Mushtaq A, Holton SJ, Schartau P, Noble ME & Sim E (20 05) Investigation of the catalytic. .. catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines Biochem J 390, 115– 123 26 Wang H, Vath GM, Gleason KJ, Hanna PE & Wagner CR (20 04) Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies Biochemistry 43, 823 4– 824 6 27 ... Biol Chem 26 3, 7 521 –7 527 22 Wang H, Guo Z, Vath GM, Wagner CR & Hanna PE (20 04) Chemical modification of hamster arylamine N-acetyltransferase 2 with isozyme-selective and nonselective N-arylbromoacetamido reagents Protein J 23 , 153–166 23 Guo Z, Vath GM, Wagner CR & Hanna PE (20 03) Arylamine N-acetyltransferases: covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline... (final concentration 320 lm) to a total 500 lL volume [26 ] The reaction rate was determined at 25 °C by measuring the linear increase in absorbance 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 represents the velocity of the hydrolysis of the acetyl-enzyme intermediate (V) The acetylated enzyme... 300 lL) The residual PABA was quantified by measuring the absorbance at 450 nm of the formation of Schiff base (e450nm = 528 35 m)1Æcm)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 (e430nm = 329 8 m)1Æcm)1) In a final volume of 300 lL, NAT2 (Y190F 0.3 lgÆmL)1, 8.76 nm; Y190I 1 lgÆmL)1, 28 nm;... 10ðpKaÀpHÞ   ¼ acetyl 1 þ 10ðpKaÀpHÞ k2 =Km ð7Þ lim À Á acetyl k2 =Km 1 þ r Á 10ðpHÀpKa2Þ   ¼ acetyl 1 þ 10ðpHÀpKa2Þ þ 10ðpKa1ÀpHÞ k2 =Km ð8Þ lim FEBS Journal 27 6 (20 09) 6 928 –6941 ª 20 09 The Authors Journal compilation ª 20 09 FEBS X Zhou et al The catalytic role of Y190 in NAT pH dependence of transacetylation of NAT with PNPA and PABA At pH values ranging from 5 .2 to 9.0, the kinetic parameters, kcat ⁄... acid by a liver-enzyme preparation Biochim Biophys Acta 65, 121 – 127 FEBS Journal 27 6 (20 09) 6 928 –6941 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 6939 The catalytic role of Y190 in NAT X Zhou et al 16 Weber WW & Cohen SN (1967) N-acetylation of drugs: isolation and properties of an N-acetyltransferase from rabbit liver Mol Pharmacol 3, 26 6 27 3 17 Riddle B & Jencks WP (1971) Acetyl-coenzyme A: arylamine. .. arylamine N-acetyltransferase Role of the acetyl-enzyme intermediate and the effects of substituents on the rate J Biol Chem 24 6, 325 0– 325 8 18 Andres HH, Kolb HJ, Schreiber RJ & Weiss L (1983) Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA: arylamine N-acetyltransferase from pigeon liver Biochim Biophys Acta 746, 193 20 1 19 Westwood IM & Sim E (20 07) Kinetic... Eqn (5), where the values of k2, Kmacetyl were obtained from presteady-state kinetics and the values of V, Etotal, and [PNPA] were from the experiments Subsequently, the half-life of the acetylenzyme intermediate, T1 ⁄ 2, was calculated from Eqn (6) acetyl Etot Km 1 1 ¼ þ þ V k2 ½PNPAŠ k2 khydrolysis ð5Þ T1 =2 ¼ Ln2=khydrolysis ð6Þ Steady-state kinetic parameters for transacetylation of arylamine substrate... (1994) Catalytic mechanism in papain family of cysteine peptidases Methods Enzymol 24 4, 486–500 Wu H, Dombrovsky L, Tempel W, Martin F, Loppnau P, Goodfellow GH, Grant DM & Plotnikov AN (20 07) Structural basis of substrate-binding specificity of human arylamine N-acetyltransferases J Biol Chem 28 2, 30189–30197 Butcher NJ, Boukouvala S, Sim E & Minchin RF (20 02) Pharmacogenetics of the arylamine N-acetyltransferases . Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue,. 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