Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2.
The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.
Dupret
J M
JM
Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Canada.
Grant
D M
DM
D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D001191:Arylamine N-Acetyltransferase