title: Chemical modification and inactivation of rat liver arginase by N-bromosuccinimide: reaction with His141.
abstract: Treatment of rat liver arginase with N-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzynska (1979) Acta Biochim. Pol. 26, 103-114). In order to probe the chemistry of N-bromosuccinimide inactivation and the role of tryptophan residues in catalysis, the two tryptophan residues of rat liver arginase, Trp122 and Trp164, have been separately mutated to phenylalanine using site-directed mutagenesis of the protein expressed in Escherichia coli. Both single Trp -> Phe mutant enzymes have kinetic parameters nearly identical to those for the wild-type enzyme. Treatment of native, wild-type, and each of the Trp -> Phe mutant enzymes with N-bromosuccinimide results in loss of absorbance at 280 nm and is accompanied by a loss of catalytic activity. However, treatment of the wild-type enzyme with N-bromosuccinimide in the presence of the arginase inhibitors NG-hydroxy-L-arginine or the combination of L-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases with N-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated by N-bromosuccinimide, indicating that His141 is the critical target for the N-bromosuccinimide inactivation of the enzyme.
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: F
authors.initials: F
authors.lastname: Daghigh
authors.forename: R C
authors.initials: RC
authors.lastname: Cavalli
authors.forename: D R
authors.initials: DR
authors.lastname: Soprano
authors.forename: D E
authors.initials: DE
authors.lastname: Ash
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001974:Bromosuccinimide / Q000494:pharmacology*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan; D014981:Xenopus
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; D001119:Arginase; D001974:Bromosuccinimide