Chemical modification and site-directed mutagenesis of human liver arginase: Evidence that the imidazole group of histidine-141 is not involved in substrate binding

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were ph otoinactivated by Rose bengal, and protection was afforded by the competiti ve inhibitor L-lysine. The dissociation constant for the enzyme-protector c omplex was essentially equal to the corresponding K-i value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wil d-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 m utation, but the mutant H141F was markedly more sensitive to trypsin inacti vation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DE PC were also insensitive to photoinactivation. We conclude that His141, whi ch is the target for both inactivating procedures, is not involved in subst rate binding, but plays a critical, albeit not essential role in the hydrol ysis of enzyme-bound substrate. (C) 1999 Academic Press.