Study of the role of the highly conserved residues Arg9 and Arg64 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 Arg64. 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 Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km 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, Arg9 and Arg64 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 Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.
Deloménie
C
C
INSERM U120, Hôpital Robert Debré, 48 boulevard Sérurier, 75019 Paris, France.
Goodfellow
G H
GH
Krishnamoorthy
R
R
Grant
D M
DM
Dupret
J M
JM
D000595:Amino Acid Sequence; D001120:Arginine; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003553:Cystine; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010658:Phenylglyoxal / Q000494:pharmacology; D011489:Protein Denaturation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D003553:Cystine; D001120:Arginine; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; C478900:NAT2 protein, human; D010658:Phenylglyoxal