Chemical Modification and Inactivation of Rat Liver Arginase byN-Bromosuccinimide: Reaction with His141

Treatment of rat liver arginase withN-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzyńska (1979)Acta Biochim. Pol.26, 103–114). In order to probe the chemistry ofN-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 inEscherichia 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 withN-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 withN-bromosuccinimide in the presence of the arginase inhibitorsNG-hydroxy-L-arginine or the combination ofL-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases withN-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated byN-bromosuccinimide, indicating that His141 is the critical target for theN-bromosuccinimide inactivation of the enzyme.