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 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.

Synthesis of inducible enzymes in irradiated yeast cells. Ultraviolet effects on transcription and the influence of recovery processes.

The induced synthesis of arginase was measured in several yeast strains after ultraviolet light irradiation. There was an exponential dose-related reduction which was the same in all cell lines tested. This sensitivity is compatible with the size of the arginase structural gene if it is assumed that one pyrimidine dimer suffices for blocking transcription. The ultraviolet-induced synthesis inhibition is susceptible to photoreactivation and liquid holding recovery. The latter process is absent in a rad2 mutant and reduced in a rad9 mutant.