Dataset Information

Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.

ABSTRACT: Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because 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, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, 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 alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic 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 superposition 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 conformation of the active site.

SUBMITTER: Zhou X

PROVIDER: S-EPMC2805756 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Publications

Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.

Zhou Xin X Zhang Naixia N Liu Li L Walters Kylie J KJ Hanna Patrick E PE Wagner Carston R CR

The FEBS journal 20091023 23

Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because 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, Tyr190, which forms a direct hydrogen bond interaction with one of the ...[more]

PMID: 19860825

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Project description:The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

Dataset Information

Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.

Publications

Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.

Similar Datasets

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