Study of the role of the highly conserved residues Arg(9) and Arg(64) in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg(64). To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg(9) and Arg(64) are the only invariant basic residues. Either mutation of the presumed catalytic Cys(68) residue or the simultaneous mutation of Arg(9) and Arg(64) to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg(9) or Arg(64) with Ala, Met, Gin or Lys exhibited unaltered K-m values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg(9)/Arg(64) With lysine in NAT1 altered the K-m for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH dependence of activity. Thus, through their positively charged side chains, Arg(9) and Arg(64) Seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg(9) and Arg(64) are involved in substrate binding and transition-state stabilization of NAT1.