L-Histidine Ammonia-lyase in Rat Liver

I. Purification and General Characteristics

Abstract

Histidine ammonia-lyase [EC 4. 3. 1. 3] was purified approximately 390 fold from female rat liver. The enzyme preparation was shown to be homogeneous and its s_{20, W} Value was 11.6 by ultracentrifugal analysis. The molecular weight was determined to be 190, 000 by the sedimentation equilibrium method.
On disc-electrophoresis, purified enzyme separated into one major and two minor components having enzyme activity, but after pretreatment with reduced glutathione, the preparation showed only one band. This suggests that the enzyme preparation was pure and that a thiol group is involved in the conversion of polymeric forms to the monomeric from. The isoelectric point was determined to be pH 5.2 by pH gradient electrophoresis.
On immunoelectrophoresis, the purified enzyme gave a single band against antihistidine ammonia-lyase serum.
The enzyme activity was highest between pH 8.8 and 9.0. The K_m value for histidine was calculated to be 1.2×10^-3_M.
Thiol compounds, such as glutathione and mercaptoethanol, considerably activated the enzyme. Parachloromercuribenzoate completely inhibited histidine ammonialyase activity at a concentration of 10^-5_M and this inhibition was partially reversed by addition of the substrate, histidine. The results indicate that a thiol is involved, not only in the conversion of the associated forms to the dissociated form, but also in the increase in catalytic activity of the enzyme.
EDTA markedly inhibited enzyme activity and this inhibition was reversed effectively by Mn²⁺, Zn²⁺, and Cd²⁺ and less effectively by Ca²⁺ and Ni²⁺. The activating effect of glutathione was lost in the presence of EDTA and reversed by addition of Mn²⁺. These results suggest that a divalent metal ion is essential for the catalytic activity of histidine ammonia-lyase and that thiol is intimately related with its function.
Sodium borohydride and nitromethane, which are known to inhibit bacterial histidine ammonia-lyase activity by reacting with dehydroalanine in the active center, considerably inhibited the enzyme from rat liver.