title: Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.
abstract: Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.
authors.affiliation: Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile. ncarvaja@udec.cl
authors.forename: N
authors.initials: N
authors.lastname: Carvajal
authors.forename: J
authors.initials: J
authors.lastname: Olate
authors.forename: M
authors.initials: M
authors.lastname: Salas
authors.forename: E
authors.initials: E
authors.lastname: Uribe
authors.forename: V
authors.initials: V
authors.lastname: López
authors.forename: P
authors.initials: P
authors.lastname: Herrera
authors.forename: J
authors.initials: J
authors.lastname: Cerpa
mesh_terms: D001119:Arginase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D001120:Arginine / Q000378:metabolism; D020134:Catalytic Domain; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000235:genetics*; D006801:Humans; D008027:Light; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000378:metabolism; D012395:Rose Bengal / Q000494:pharmacology
chemical_list: D012395:Rose Bengal; D006639:Histidine; D001120:Arginine; D009952:Ornithine; D001119:Arginase; D008239:Lysine; D004047:Diethyl Pyrocarbonate