MEDI 67 |
| Arylamine N-acetyl transferases (NATs) are responsible for detoxification of arylamine and arylhydrazine drugs and potentiation of carcinogenic xenobiotics through transferring an acetyl group from acetyl CoA to various substrates. The active site cysteine of hamster NAT2 has been shown to be the highly reactive thiolate-imidazolium ion pair with a pKa of 5.2. 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, which is dependent on a shift in the catalytic triad histidine pKa from >9 to 5.5. Beyond the obvious contributions of the Asp-122, the parameters governing this remarkable change are not understood. Upon closer inspection of the bacterial NAT structure, the conserved residue, Tyr-190, was found to form a close hydrogen bond with Asp-122. To assess the importance of this residue on the catalytic mechanism of NAT's we prepared a series of mutants at this position to determine the role of the hydroxyl moiety (Tyr-190 to Phe), hydrophobicity (Tyr-190 to Isoleucine) and side-chain packing (Tyr-190 to Alanine). The Y190F, Y190I, and Y190A mutants exhibited significantly reduced kcat values for transacetylation of p-aminobenzoic acid (PABA) from the acetyl donor p-nitrophenylacetate (PNPA) compared to wild type. For both the Y190I and Y190A mutants, pre-steady state and steady-state kinetic analyses revealed elevated pKa's for the pH vs. rate data (approximately 0.5-1 unit) either in the first step (acetylation of the NAT) or in the second step (transacetylation of the arylamine substrate), resulting in a much less reactive active site cysteine and altered acceptor substrate specificity. Collectively, these results reveal that tyrosine 190 is intimately involved in maintaining the electrostatic potential of the hamster NAT2, and likely human NAT1, catalytic residues. |
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Poster Session
7:00 PM-9:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster
Sci-Mix
Division of Medicinal Chemistry |