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Arylamine N-acetyltransferases (NATs) play an important role in both detoxification of arylamine and hydrazine drugs and activation of arylamine carcinogens. Since the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr-190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp-122, as well as a potential pi-pi stacking interaction with the active site His-107. Replacement of Hamster NAT2 Tyr-190 by either phenylalanine, isoleucine, or alanine was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr-190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid (PABA) when compared to wild type. Tyr-190 was also shown to play an important role in determining the pKa of the active site cysteine during acetylation, as well as the pH versus 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 superposition of [1H, 15N] HSQC spectra for wild type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr-190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of active site.
Arylamine N-acetyl transferases (NATs,1 EC 126.96.36.199) are ubiquitous enzymes in nature that catalyze N-acetylation of arylamines and O-acetylation of arylhydroxylamines, as well as N,O-transacetylation of arylhydroxamic acids . These reactions result in detoxification of arylamine and arylhydrazine drugs, such as isoniazid, sulfonamides, procainamide, and hydralazine, reducing the potential for CYP-dependent N-oxidation [2, 3], which is also responsible for bioactivation of arylamine environmental toxicants, such as 2-aminofluorene (2-AF), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo(4,5-b)-pyridine (PhIP) [4, 5]. Humans express two NAT isozymes (NAT1 and NAT2) that have 81% sequence identity but differ in substrate specificity and tissue distribution [6–8]. Human NAT2, which is found mainly in the liver  and the intestine , selectively acetylates substrates such as isoniazid (INH), sulfamethazine (SMZ), daspone, and procainamide , whereas human NAT1, which is extensively distributed and expressed early in development at the blastocyst stage , preferentially acetylates substrates such as p-aminobenzoic acid (PABA), p-aminosalicylic acid (PAS), and p-aminobenzoyl-glutamic acid (pABglu) [6, 12]. The widespread expression of human NAT1 and the selectivity for p-ABglu, as well as the presence in blastocytes and fetal tissues of NAT 1, has suggested that this enzyme may have a role in folate metabolism and neural tube development [13, 14].
Since many human NAT substrates are carcinogens 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] AcCoA in the absence of amine  and the active site Cys68 or Cys69 has been further identified through thiol-specific modification and site-directed mutagenesis [22–24]. The first crystal structure of NAT, from Salmonella typhimurium (StNAT) (PDB code: 1E2T), revealed a strictly conserved Cys-His-Asp catalytic triad, reminiscent of cysteine proteases . Site-directed mutagenesis experiments with NATs have confirmed that each residue of the triad is individually essential for catalysis and protein stability [24–26].
Our laboratory has investigated 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 analogy all NATs, proceeds through rapid formation of an acyl-cysteine intermediate, followed by rate limiting acyl transfer . The exceptional reactivity of the active site cysteine 68 can be attributed to the formation of a thiolate-imidazolium ion pair with a pKa of 5.2 . However, in contrast to cysteine proteases, which typically exhibit an additional basic limb pKa of 8–9, the second pKa for hamster NAT2 acetylation, was found to be >9.5. For both NATs and cysteine proteases, the basic pKa has been attributed to the triad histidine [26, 32].
Elucidation of the influence of His-107 and Asp-122 on the catalytic reactivity of Cys-68 has remained elusive, since mutations at these two positions (e.g. D122N, D122A, H107Q, H107N) generate insoluble protein with no detectable activity even after refolding [26, 27]. Consequently, we hypothesized that modulation of the catalytic potential of the catalytic triad might be accessible through point site mutations of the proximal residue, tyrosine 190, which is highly conserved and participates in hydrogen bonding with Asp-122 (2.6 angstroms in StNAT crystal structure (PDB code: 1E2T) , 2.8 angstroms in human NAT1 crystal structure (PDB code: 2QPT) , and 3.28 angstroms in hamster NAT2 NMR model structure (PDB code: 2GWY) ), as well as potential Π–Π interactions with His-107 (Figure 1). This tyrosine is highly conserved [34, 38, 39] in all the NAT sequences reported to date, with the only exception being the isoform banatB from Bacillus anthracis, where a histidine is at the equivalent position . In addition, there is an array of known NAT polymorphisms of which some have been associated with an increased cancer risk . Generally these mutations have resulted in loss of NAT activity due to either catalytic triad mutations, decreased enzyme stability or sequence truncation . We hypothesized that unlike other active site residues, mutations at Tyr-190 might be tolerated, despite its conservation, since inactive NAT polymorphisms at this position have not been identified . Furthermore, active genetic variants at position 190 have been identified by chemical mutagenesis .
Consequently, we carried out steady-state and transient state kinetic studies on a series of mutants at this position to delineate the contribution of the hydroxyl moiety (Tyr-190 to Phe), aromatic stacking (Tyr-190 to Ile), and interior side-chain packing (Tyr-190 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.
Similar circular dichroism spectra were observed for wild-type and Y190 mutants at pH 7 (See Supplemental Information Figure. S1), which further confirmed that the Y190 mutations, unlike the H107 and D122 mutations, did not disrupt the overall secondary structure composition of the protein [26, 27]. To probe the structural implications of Y190 mutations more deeply, we used [1H, 15N] HSQC experiments to record the chemical shift values of NAT amide nitrogen and hydrogen atoms. 15N-labeled proteins were prepared and [1H, 15N] HSQC experiments were carried out, and the resulting spectra collected at 600 MHz were superimposed. Consistent with the CD spectra, the amide resonances of most of the residues in secondary structural elements were unperturbed; however, the Y190A mutation caused nearly all of the amides of residues in the catalytic cavity to shift (SI. Fig. S2A). Such shifting is caused by changes in the atom’s chemical environment, and the affected residues include those proximal to Y190, such as H107, D122, F125, and F192, the latter of which forms an edge-to-face aromatic stacking with Y190 (SI. Fig. S2B). Also included, however, are L69, S224, and F288, which are up to 18Å 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 tyrosine 190 disturbs the conformation of these catalytic triad residues . In addition, residues close to D122 (I120, V121, A123, and G124), and residue L69, close to C68, and residue L108, close to H107, have been affected. Changed chemical shifts of F125 and F192, which form the edge-to-face aromatic stacking with Y190, were also observed (SI. Fig. S2B). In addition to the observed shifting, residue attenuation is observed, most obviously for L69, L108, D122, and A123. Such attenuation is caused by chemical exchange and suggests that the catalytic cavity configuration compensates for the loss of Y190.
Since 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 saturating PNPA concentrations and fixed PABA and NAT2 enzyme concentrations. The measured activities were 184± 8 μmol/mg/min, 130± 21 μmol/mg/min, 22± 3 μmol/mg/min, and 8.5± 0.7 μmol/mg/min for wild-type, Y190F, Y190I, and Y190A, respectively. Therefore, under the given conditions, eliminating the hydroxyl group of Tyr 190 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.
The rate of acetylation of NATs was determined with a stopped flow apparatus by measuring the fast release of PNP before the acetylated enzyme concentration reaches steady state. Each of the Y190 mutants demonstrated similar “burst kinetics” as observed for wild type , 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 wild-type (Table 1), 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-fold and 87-fold, respectively. Consequently, Y190 appears to be necessary for maintaining the optimal reactivity of the cysteine 68 for acetylation.
As previously demonstrated by single turnover kinetics with PNPA, acetylation of wild type NAT proceeds through rapid formation of an enzyme intermediate (k2) followed by rate-limiting hydrolysis (khydrolysis)  (Scheme 1). Each of the Y190 mutants exhibited similar burst kinetics, followed by rate-limiting deacetylation by water (Table 1). Nevertheless, the rate of hydrolysis (khydrolysis) for each mutant was found to have significantly increased by 4-to 30- fold, relative to wild-type, resulting in a 3.5- to 40- fold decrease in the life-time of the acetylated enzyme. Removal of the para-hydroxyl group by the Tyr-190 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 intermediate hydrolysis (khydrolysis) of 3.6-fold. Similarly, a decrease in k2 and increase in khydrolysis (about 29-fold) was observed when the phenol moiety of Tyr-190 was replaced by the sec-butyl group of isoleucine. The Tyr-190 to Ile mutation resulted in the largest decrease in acetylated enzyme stability. When the tyrosine 190 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, while 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 acetylation and deacetylation, perturbation of the active site by complete removal of the side chain mainly affected enzyme acetylation (k2).
Usually, the pH dependence of acetylation of NAT (k2/Kmaceytl) reflects ionizations of the free enzyme and free substrate that are either directly or indirectly involved in substrate binding and in the catalytic process . For wild-type, the pH dependence of the single turnover rate constant, k2/Kmacetyl, fit best to a model for two pKas with the first pKa1acetyl (5.16± 0.14) assigned to the active site cysteine, and the second pKa2acetyl (6.79± 0.25) assigned to a probable conformational change . 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 (Figure 2). This suggests that removal of the hydroxyl group from Y190 results in little perturbation of the active site cysteine 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 is 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 conformational change at the active site. The more drastic mutations, Y190I and Y190A, however, revealed the importance of the phenyl ring of the tyrosine 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, compared to wild type 5.16± 0.14). Thus, the reactivity of the catalytic cysteine was reduced for these two mutants, which is consistent with the significant decrease in the observed rate of acetylation.
The pH dependence of transacetylation of PNPA/PABA (kcat/KPABA) reflects 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 step. 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−1s−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 . 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 (Figure 3). In our previous solvent isotope effect study of wild-type NAT2, a normal SKIE (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 His-107 was identified as the likely base with a pKatransacetyl of 5.55 for the acetylated enzyme . We assumed general base catalysis was also employed by the Y190 mutants, thus, the first pKa1transacetyl values from the fitting results were assigned to the His-107 for the acetylated mutant NAT2. Accordingly, the transacetylation of PNPA/PABA by Y190F proceeds with a pKa1transaccetyl of 5.48± 0.06, which is similar to the pKatransacetyl of 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 significantly lower transacetylation rates and thus, the overall importance of Y190 on the protonation state of His-107.
The transacetylation of arylamine (k4) (Scheme 2) from acetylated NAT2 proceeds much faster (1000 to 10000 fold) than hydrolysis of the acetylated NAT intermediate (khydrolysis)  (Scheme 1). Using PNPA or AcCoA as the acetyl donor and PABA, anisidine, pABglu or 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 reactions with PNPA as the acetyl donor, the transacetylation of arylamine substrate (k4), rather than the acetylation of NAT2 (k2), is the rate limiting step . Therefore, the kcat values for PNPA/anisidine, PNPA/PABA, PNPA/pABglu approximate k4 (the rate of transacetylation of amine acceptors) (Equation 9). However, for reactions employing AcCoA as the acetyl donor, the kcat values are determined by the individual rate constants for both the acetylation (k2) and deacetylation (k4) steps (Equation 10) . Based on the ping-pong mechanism, the acetylation rate (k2) of NAT2 by the acetyl donor is independent of the transacetylation 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 (Equation 10), the values of k2 for AcCoA can be predicted to range from 10–1740 s−1. Hence, from the kcat values for the acetylation of PNA by AcCoA, we are able to predict the k4 values for PNA to be 0.60s−1, 0.31s−1, 0.89s−1, 0.26s−1, for wild type, Y190F, Y190I, and Y190A, respectively. These k4 values are similar to the kcat values, indicating that in contrast to the acetylation of PABA by AcCoA, deacetylation (k4) is the rate limiting step when PNA is the acetyl acceptor.
Since the arylamine substrates possess different pKas, we further quantified the effect of the substrate’s pKa on k4 by constructing a Brønsted plot. This is shown in Figure 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 pKas 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) . 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 histidine . In contrast to wild-type NAT2, the Brønsted plot for Y190I, Y190A and Y190F clearly demonstrated an altered dependence of the reaction on the pKa and nucleophilicity of the acceptor amine. Smaller βnuc values were observed (βnuc = 0.6 ± 0.1, for Y190F, βnuc = 0.4± 0.1, for Y190I, and βnuc = 0.5 ± 0.1, for Y190A) for the Brønsted plot for the pKNH3+ (or pKH3O+) ranging from −1.7 to 5.34 (Figure 4). These results indicate that for the mutants less proton transfer occurs during the transition state as compared to the wild type, and there is less bond formation between the nitrogen and the thioester carbonyl than occurs for wild type. In addition, since the increase in the pKatransacetyl for Y190I and Y190A approximates the anisidine pKa, the reaction shifts for these mutants from being dominated by the deprotonation of a tetrahedral intermediate (Scheme 3, TS-II) to nucleophilic attack of the thioester (Scheme 3, TS-I).
For anisidine, deprotonation must occur by Y190F after formation of the tetrahedral intermediate, since the pKatransacetyl for His-107 (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 prior to formation of the tetrahedral intermediate.
The essential Cys-His-Asp catalytic triad in arylamine N-acetyltransferase has been identified among several prokaryotic and eukaryotic 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 . In contrast, the unavailability of active mutants of hamster NAT2 at His107 and Asp122 after refolding suggested these two catalytic residues have both catalytic and structural 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 crystallographic analysis revealed that the para hydroxyl moiety of Tyr-190, which resides at a β sheet that is close to the 17-residue insertion loop (163–187) (Figure. 1A), is positioned within the active site hydrophobic core, where the hydroxyl group forms a hydrogen bond with aspartate 122 of the catalytic triad (Figure 1). This tyrosine 190 is highly conserved across prokaryotic and eukaryotic NATs [34, 38], with the only exception being the truncated banatB isoform from Bacillus anthracis, where a histidine is at the equivalent position . Closer inspection revealed that in addition to the side chain of Tyr-190, the side chain of Asn-72 and the backbone of Gly-124 and Ala-123 participate in a network of interactions with Asp-122. Moreover, since the centroid of the Tyr-190 phenyl ring is approximately 3.5 from the centroid of the His-107 imidazole ring and the planes of the two ring systems intersect at an angle of about 30°, Tyr-190 and His-107 interact by a common aromatic stacking interaction. To gain insight into the role of Tyr-190 on NAT catalysis, we characterized a set of point site mutants at this position by steady-state and pre-steady state kinetics and NMR spectroscopy.
Unlike His107 and Asp122, mutations at the 190 position in hamster NAT2 neither affect the protein’s overall folding and stability nor abolish the enzymatic activity, indicating 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 , while the Tyr-to-Ile substitution is expected to maintain the secondary structure, since β strand formation is favored by isoleucine . 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, since replacement of a phenol side chain with a methyl group eliminates hydrophobic packing interactions 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 Tyr-190 to Phe mutant and wild type. However, the rate of acetylation of NAT2 (k2) is 5-fold lower than wild type, and the stability of the acetylated enzyme intermediate is affected, which can be attributed to the removal of the hydrogen bond between the tyrosine hydroxyl group and the aspartyl carbonyl group (Table 1). Therefore, the modest decrease in the kcat values for transacetylation of PNPA/PABA suggests that the role of the hydroxyl group (i.e. H-bonding) of Tyr-190 in hamster Y190F is masked by the turnover number, which is mainly affected by k4 rather than k2. In contrast, the significant loss of catalytic efficiency for the Y190I and Y190A mutants is supportive of a potential role in catalysis played by the imidazole-aromatic interaction between the Y190 and H107 (Figure 1) [41, 42]. Loewenthal et al have found that the aromatic-histidine interaction in barnase stabilizes the protonated histidine, increasing its pKa value and, therefore, increasing the nucleophilicity of active site cysteine . A similar interaction was found between the indole ring of an active site tryptophan 177 and the imidazole of the catalytic triad histidine 159 in the papain-like cysteine proteinase . Mutations of the tryptophan to either tyrosine, phenylalanine, isoleucine or alanine (the strength of the histidine-aromatic interaction decreases in the series His-Trp greater than His-Tyr greater than His-Phe) lead to elevation of the cysteine pKa and destabilization of the thiolate-imidazolium ion pair . Similarly, pKa1acetyl, which has been associated with Cys-68 for the hamster NAT2 , was raised by approximately one pKa unit when Tyr-190 was replaced with the aliphatic amino acid, isoleucine or alanine.
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 wild type; nevertheless, the first inflection point is similar, corresponding to the pKaacetyl of the active site cysteine. This unchanged pKaacetyl of the active site cysteine in Y190F could be ascribed to dipole-dipole interaction between the para hydrogen of phenylalanine and the aspartyl oxygen that stabilizes the formation of the thiolate-imidazolium ion pair through Asp122, albeit less efficiently than the tyrosine hydroxyl [45, 46]. Second, it is problematic to assign the second pKaacetyl for acetylation of the wild type (pKa2acetyl 6.79±0.25) to the ionization of the OH group in Y190. It is tempting to assign this pKa2acetyl to Y190, since 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 the pKa2acetyl reflects ionization of a pH-sensitive residue that indirectly affects conformation of the active site since no putative ionizable side chain responsible for this pKa2acetyl appears in the active site. The lower reactivity of the active site cysteine in Y190I and Y190A is consistent with the elevated pKa1acetyl from k2/Kmacetyl versus pH since these side chains are likely to raise the pKa of Asp-122 and thus Cys-68.
While considerably different from the wild type profile, the pH rate profiles for transacetylation for three mutants with PNPA/PABA were similar to each other. The pKa1transacetyl of Y190F is similar to wild type, while the pKa1transacetyl values of Y190I and Y190A are about one unit higher. Under the assumption that, like wild type NAT2, the Y190 mutants utilize general base catalysis and His 107 corresponds to the first pKa1transacetyl, the experimental data are consistent with our previously proposed model . The pKa1transacetyl 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 pKa1transacetyl of the active site histidine (5.55± 0.14) is matched to that of PABA (pKa = 4.67), thus facilitating concerted deprotonation of the incoming arylamine nucleophile in the transition state (Scheme 3, TS-I). If, however, the pKatransacetyl of the histidine is significantly lower than that of the conjugate acid of the attacking arylamine, then deprotonation is favored to follow the tetrahedral intermediate 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) since 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, since it would be expected that the altered pKa1transacetyl of active site histidine would be matched by that of the associated altered Asp-122. To address this issue we carried out protein NMR structural studies of the most altered mutant, Y190A. The results of those studies revealed an altered active site, including Asp-122 and the most closely associated inner sphere side chains. Consequently, we propose that Y190 is likely to function not only as a hydrogen bond donor to Asp-122, but also as a “damper” of the inherent sensitivity of the active site to undergo re-organization. Recently the importance of protein dynamics on catalysis has become increasingly apparent [47, 48]. The backbone dynamics of hamster NAT2 has been characterized by NMR experiments, with slower, low frequency motions, detected for the active site cavity . 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” structure to accommodate substrate interaction . 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 truncations, the availability of the Tyr 190 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 bioactivation of N-arylhydroxylamines by O-acetylation to produce DNA adducts, and on the intracellular fate of the NAT protein [50, 51].
AcCoA, PABA, PNPA, ampicillin, anisidine, MOPS, 3,3-dimethylglutaric acid, pABglu, and PNA were purchased from Sigma-Aldrich (St. Louis, MO). BL21 Codon Plus (RIL) competent Escherichia coli cells were purchased from Stratagene (La Jolla, CA). DEAE Sepharose Fast Flow anion-exchange resin was purchased from Amersham Pharmacia (Ann Arbor, MI). Steady state kinetic data were collected on a Varian Cary 50 UV-Vis spectrophotometer (Palo Alto, CA). Transient kinetic data were obtained on a single-wavelength stopped-flow apparatus (Applied Photophysics, model SX.18MV). Kinetic data were analyzed with the JMP IN 4 software (SAS Institute, Inc.)
Site-directed mutagenesis of the hamster NAT2 Tyr-190 to Phe (Y190F), to Ile (Y190I) and to Ala (Y190A) were carried out by using the pPH70D vector and QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotide primers used for Y190F, Y190I, and Y190A were 5′-GA AAG ATC TTT190 TCT TTT ACT CTT GAA CCC CG - 3′, 5′-GA AAG ATC ATT190 TCT TTT ACT CTT GAA CCC CG - 3′, and 5′-GA AAG ATC GCT190 TCT TTT ACT CTT GAA CCC CG – 3′, respectively. The automated DNA sequencing results showed the desired sites of mutations have been achieved. The mutated plasmids were transformed to BL-21 Codon Plus (RIL) E. coli cells according to the protocol of the manufacturer.
The expression and purification of the mutants was similar to those for wild-type hamster NAT2 as previously described . Overnight cultures (10 ml) were grown from single colonies and were diluted to 1 liter of Terrific Broth (TB) containing ampicillin (100ug/ml) and chloramphenicol (50ug/ml). Cultures were grown at 37°C to an OD600 of 0.6, at which time IPTG was added to a final concentration of 0.2mM. After IPTG induction, cells were incubated for additional 17 hours of growth at 17°C and harvested. The cell pellets were lysed as previously reported . The mutated NAT2-dihydrofolate reductase (DHFR) fusion proteins were purified by an ion exchange column (50mm diameter) packed with Q-Sepharose fast flow beads (Pharmacia, 60ml) and eluted from the column at 0.26M KCl. The DHFR-NAT2 fusion proteins subsequently underwent human thrombin cleavage and applied to the second Q-Sepharose column. NAT2 was eluted at 0.08M KCl. Both columns were coupled with a Pharmacia FPLC system with an LCC 500 plus system controller, two P500 solvent delivery pumps, and a P500 collector. Protein concentrations were determined with the Bradford protein assay .
The specific activity of wild-type and mutant NAT2 was measured using p-nitro phenyl acetate (PNPA) as acetyl donor and p-amino benzoic acid (PABA) as acetyl acceptor in MOS buffer (pH7, 25°C) as described previously . The reaction buffer contains 0.5 μg/ml of NAT2, 0.5 mM of PABA and the reaction was initiated by adding PNPA in DMSO (final concentration 2 mM, DMSO 1%). The rate of the reaction was determined by monitoring the linear increase of absorbance at 400 nm because of the formation of p-nitro phenol. The specific activity was calculated and expressed in μM/mg/min.
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 (160–3000μM) in MOPS buffer [1ml, 100mM; 150mM NaCl, and 3% DMSO (pH 7.0)] was transferred to one stopped flow syringe. NAT2 (Y190F, 276ug/ml, 8μM; Y190I 353ug/ml, 10.2μM; Y190A 642ug/ml, 18.6μM) in MOPS buffer [1ml, 100mM; with 150mM of NaCl (pH 7.0)] was transferred to the second stopped flow syringe. Each time equal volumes (50μL) of the enzyme solution and the substrate were injected and mixed rapidly. The production of p-nitro-phenol [P] was monitored at 400nm . The single turnover time-course curves were fitted with equation 1 using JMP IN 7 software, where A is the amplitude and kobs is the pseudo-first-order rate constant for the acetylation step. The results represent the average of three experiments. The kinetic parameter k2/Kmacetyl was obtained by plotting kobs versus PNPA concentration (equation 2) (Fig. S3).
Hamster NAT2 (0 μM, 1 μM, 2 μM, 4 μM, and 8 μM) in MOPS buffer [100 mM; with 150 mM of NaCl (pH 7.0) and 0.1 mM DTT] was added with PNPA (final concentration of 320 μM) to a total 500 μL volume . The reaction rate was determined at 25°C by measuring the linear increase of absorbance at 400nm during the initial 5 min. Control experiments were carried out in the absence of the NAT2. The slope of the linear increase of Abs400nm represents the velocity of the hydrolysis of the acetyl-enzyme intermediate (V). The acetylated enzyme hydrolysis rate constant, khydrolysis, can be calculated by equation 3, where the values of k2, Kmacetyl were obtained from pre-steady state kinetics and the values of V, [E]total, and [PNPA] were from the experiments. Subsequently, the half life of the acetyl-enzyme intermediate, T1/2, was calculated from equation 4.
As described previously, the assay was performed using PNPA or AcCoA as the acetyl donor and one of the following primary arylamines as the acetyl acceptor: anisidine, PABA, pABglu, or PNA . The reaction rates were determined in triplicate at 25°C at a fixed concentration of one substrate while varying concentration of the other substrate. All the kinetic parameters were determined with at least five different concentrations of each substrate.
The initial velocities of the reaction with PNPA/anisidine, PNPA/PABA, and PNPA/pABglu were measured as the linear increase in the absorbance at 400nm due to the formation of PNP (ε400nm = 9400 M−1 cm−1). In a final volume of 500 μL, NAT2 (Y190F 0.3μg/ml, 8.76nM; Y190I 1μg/ml, 28nM; Y190A 3μg/ml, 88nM) was incubated with either anisidine (0.025–1.6mM), PABA (0.05–1.2mM), pABglu (0.25–12mM) or PNA (0.5–8mM) in MOPS buffer (100 mM at pH 7.0, 150mM NaCl, and 0.1mM DTT). The reactions were initiated by addition of PNPA dissolved in DMSO (20 μL). The final concentration of DMSO was 4%.
The initial velocity of the reaction with AcCoA/PABA was measured as a linear decrease of PABA concentration. In a final volume of 1000μL of dimethylglutaric acid buffer (50 mM, 80 mM NaCl, and 0.1 mM DTT at pH 7.0), NAT2 (Y190F 0.3μg/ml, 8.76nM; Y190I 0.5μg/ml, 14nM; Y190A 1.5 μg/ml, 44 nM) was incubated with AcCoA(0.125–8 mM) for about 1 min, then the reaction was initiated by adding PABA (0.03–0.4mM). Aliquots (20–120 μL) of the reaction mixture were withdrawn at 10s–20s intervals (0–200s) and transferred to an assay mixture containing TCA (4%, v/v), DMAB (2.5%, w/v), and acetonitrile (45%, v/v) (final volume of 300 μL). The residual PABA was quantified by measuring the absorbance at 450 nm of the formation of Schiff base (ε450nm = 52835 M−1 cm−1) .
The initial velocity of the reaction with AcCoA/PNA was measured as a linear decrease in the absorbance at 430nm because of the acetylation of PNA (ε430nm =3298 M−1 cm−1). In a final volume of 300μL, NAT2 (Y190F 0.3μg/ml, 8.76nM; Y190I 1μg/ml, 28nM; Y190A 3μg/ml, 88nM) was incubated with AcCoA (10μL, final concentration 0.04–0.8mM) and PNA (0.05–2.5mM) in dimethylglutaric acid buffer (50 mM, 80 mM NaCl, and 0.1 mM DTT at pH 7.0). The reactions were monitored over a maximum of 5 min.
At pH values ranging from 5.2–9.0, the kinetic parameter, k2/Kmacetyl, was determined using stopped-flow apparatus with NAT2 (final concentration Y190F 4μM, Y190I 10μM, Y190A 19μM) and PNPA (final concentration of 800 μM). Either dimethylglutaric acid buffer (50mM at pH 5.0–7.0, 0.1mM DTT) or Tris buffer (50mM at Ph 7.5–9.0, 0.1mM DTT) was used. The ionic strength was held constant at 150mM with NaCl. Assays were performed in triplicate as described for the pre-steady state kinetic experiments (vide supra), except that the formation of p-nitrophenol was monitored at 340 nm for pH 5.2–6, and at 400 nm for pH 6.4–9. The single turnover time-course curves were fitted with equation 1 using JMP IN 4 software, where the pseudo-first-order rate constant, kobs, for the acetylation step, was abstracted, and the parameter k2/Kmacetyl was obtained. To fit the k2/Kmacetyl versus pH data for wild-type, Y190I, and Y190A, equation 6 was used, and for Y190F, equation 5 was used.
At pH values ranging from 5.2–9.0, the kinetic parameters, kcat/KPABA, was determined at a fixed, saturated concentration of PNPA (2mM) and six concentrations of PABA (10–120mM) with NAT2. Either dimethylglutaric acid buffer (50mM at pH 5.0–7.0, 0.1mM DTT) or Tris buffer (50mM at Ph 7.5–9.0, 0.1mM DTT) was used. The ionic strength was held constant at 150mM with NaCl. Assays were performed as described in previous steady state kinetics, except that the formation of p-nitrophenol was monitored at the isosbestic point of 349 nm (ε349nm = 5700 M−1 cm−1). To fit the k2/KPABA versus pH data for wild-type, equation 7 was used, and for Y190F, Y190I, and Y190A, equation 8 was used.
For CD spectroscopy, 3μM of NAT2 protein samples were dissolved in 10mM potassium phosphate (pH 7) containing 0.5mM EDTA and 0.05mM DTT. Spectra were acquired at 25°C on a Jasco J-710 spectropolarimeter. For HSQC experiments, 15N-labeled wild-type and Y190A hamster NAT2 were prepared by growth in M9 minimal medium with 15N-labeled ammonium chloride (1g/liter M9 minimal medium) as the only nitrogen source [34, 52]. The purified 15N-labeled protein was concentrated to 0.2 mM in NMR buffer (30 mM of sodium phosphate, 50 mM of NaCl, 4 mM of DTT, pH 6.8, 0.1% (w/v) NaN3, 10% D2O). HSQC spectra were acquired on Varian 600MHz spectrometers equipped with a Bioselect probe at 10 °C as described .
Figure S1: Circular Dichroism of wild type and Y190 mutants at pH 7 at room temperature
Figure S2: (A) Superposition of [1H, 15N] HSQC spectra of wild type hamster NAT2 (black) and Y190A-hamster NAT2 (red). (B) Affected residues in Y190A hamster NAT2.
Figure S3: Pre-steady state kinetics of formation of the acetyl-enzyme intermediate by wild type and Y190 mutants.
This work was funded by grants from the Leukemia Research Foundation (CRW), Developmental grants for Drug Design, the department of medicinal chemistry, 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.