Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by <f>Mn2+</f>. The effect was reversed by hydroxylamine and involved a residue with a <f>pKa</f> of <f>6.5±0.1</f>. Half activation with <f>Mn2+</f> was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in <f>Km</f> for arginine or <f>Ki</f> for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added <f>Mn2+</f> and contained <f><0.1</f> <f>Mn2+</f>/subunit, whereas H101N was half active and contained <f>1.2±0.1</f> <f>Mn2+</f>/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state. [Copyright &y& Elsevier]