Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis.
Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.
Ito
M
M
Division of Molecular Genetics, Fujita Health University, School of Medicine, Aichi, Japan.
Kato
S
S
Nakamura
M
M
Go
M
M
Takagi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D014503:Urate Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019820:Xanthine; D014970:Xanthines / Q000494:pharmacology
D014970:Xanthines; D019820:Xanthine; D014503:Urate Oxidase