Catalytic mechanism of hamster arylamine N-acetyltransferase 2

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Br?nsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.     

Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.     

A density functional theory study on the role of His-107 in arylamine N-acetyltransferase 2 acetylation

Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to primary arylamines, and are responsible for the biotransformation and metabolism of drugs, carcinogens, etc. Structure analysis revealed that His-107 was likely the residue accountable for mediating acetyl transfer. We have examined the full catalytic mechanism of this system by means of DFT method. The results indicate that if the acetyl group directly transferred from the donor, p-nitrophenyl acetate, to the acceptor, cysteine, the high activation energy will be a great hindrance. These energies have dropped a little in a range of 20-25 kJ/mol when His-107 is assisting the transfer process. However, when protonated His-107 is mediating the reaction, the activation energies have dropped about 70-85 kJ/mol. Our calculations strongly support an enzymatic acetylation mechanism that experiences a thiolate-imidazolium pair, which have verified the presumption from experiments.     

Peroxynitrite irreversibly inactivates the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) in human breast cancer cells: a cellular and mechanistic study

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.     

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis

Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in mammalian NATs, possess inherent motions on the nanosecond time scale. The latter segment becomes more restricted in its motions upon substrate binding according to our NMR XNOE data. This greater rigidity appears to stem from interactions with the substrate. Finally, NAT acetylation has been suggested to protect these enzymes from ubiquitination. Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection.     

The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity.

Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms. NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans. The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases. The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed. To investigate the function of the first two domains of full-length NAT from S. typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing. Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S. typhimurium are toxic when overexpressed intracellularly in Escherichia coli. Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate. Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S. typhimurium.     

The unusual catalytic triad of poliovirus protease 3C

Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.     

An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although prokaryotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins.     

Composition and function of the eukaryotic N-terminal acetyltransferase subunits

Saccharomyces cerevisiae contains three N-terminal acetyltransferases (NATs), NatA, NatB, and NatC, composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p (NatA); Nat3p and Mdm20p (NatB); and Mak3p, Mak10, and Mak31p (NatC). The overall patterns of N-terminally acetylated proteins and NAT orthologous genes suggest that yeast and higher eukaryotes have similar systems for N-terminal acetylation. The differential expression of certain NAT subunits during development or in carcinomas of higher eukaryotes suggests that the NATs are more highly expressed in cells undergoing rapid protein synthesis. Although Mak3p is functionally the same in yeast and plants, findings with TE2 (a human Ard1p ortholog) and Tbdn100 (a mouse Nat1p ortholog) suggest that certain of the NAT subunits may have functions other than their role in NATs or that these orthologs are not functionally equivalent. Thus, the vertebrate NATs remain to be definitively identified, and, furthermore, it remains to be seen if any of the yeast NATs contribute to other functions.     

Denatured state thermodynamics: residual structure, chain stiffness and scaling factors

A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum histidine-heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37-83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2+/-0.3. This value is much larger than the scaling factor of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins.     

Impairment of the activity of the xenobiotic-metabolizing enzymes arylamine N-acetyltransferases 1 and 2 (NAT1/NAT2) by peroxynitrite in mouse skeletal muscle cells

Reactive nitrogen species and their by-products, such as peroxynitrite, modulate many physiological functions of skeletal muscle. Peroxynitrite generation occuring under specific conditions, such as inflammation, may also lead to skeletal muscle dysfunction and pathologies. Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes (XMEs) involved in the detoxification and/or metabolic activation of several drugs and chemicals. In addition to other XMEs, such as gluthatione S-transferases or cytochromes P450, NAT enzymes are expressed in skeletal muscle. We show here that functional NAT1 and NAT2 isoforms are expressed in mouse myotubes and that peroxynitrite may impair their activity in these cells. We show that this inactivation is likely due to the irreversible modification of NATs catalytic cysteine residue in vivo. Our results suggest that peroxynitrite-dependent inactivation of muscle XMEs such as NATs may contribute to muscle dysfunction by impairing the biotransformation activity of this key cellular defense enzyme system.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the front-line antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of M. tuberculosis NAT which reveals that substituted anilines are excellent substrates but that isoniazid is a very poor substrate for this enzyme. We propose that the expression of NAT from M. tuberculosis (TBNAT) is unlikely to be a significant cause of isoniazid resistance. The kinetic parameters for a variety of TBNAT substrates were examined, including 3-amino-4-hydroxybenzoic acid and AcCoA, revealing K m values of 0.32 +/- 0.03 and 0.14 +/- 0.02 mM, respectively. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a bi-bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters reveals that one enzyme group must be deprotonated for optimal catalytic activity and that two amino acid residues at the active site of the free enzyme are involved in binding and/or catalysis. Solvent kinetic isotope effects suggest that proton transfer steps are not rate-limiting in the overall reaction for substituted aniline substrates but become rate-limiting when poor hydrazide substrates are used.     

The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the “mammalian insertion”, a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.     

The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.     

Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) > 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.